Chimeric cytokine formulations for ocular delivery

ABSTRACT

Featured herein are vehicle formulations and formulations containing a chimeric cytokine designed for e.g., ocular delivery.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/779,974, filed Mar. 13, 2013, the entire content of which is herebyincorporated in its entirety.

FIELD OF THE INVENTION

The present invention relates to therapeutic compositions andformulations, e.g., for IL-1 inhibitors.

BACKGROUND

Interleukin-1 alpha (IL-1α) and beta (IL-1β) are members of the IL-1family of immunoregulatory cytokines. At least eleven human members ofthe interleukin-1 cytokine family have been identified, nine putative ordemonstrated agonists (IL-1α, IL-1β, IL-18, IL-36α (IL-1F6), IL-36β(IL-1F8), IL-36γ (IL-1F9), IL-33, IL-1F7 and IL-1F10) and two naturalantagonists (IL-1Ra and IL36Ra (IL-1F5)).

IL-1α and IL-1β have roles in regulating the immune system, and havebeen implicated in inflammatory ophthalmic diseases, includingsignificant inflammatory ocular disorders. Thus, there is a need forbetter methods and materials for ocular administration of IL-1inhibitory agents.

Anakinra (Kineret®, Amgen, Thousand Oaks, Calif.), a recombinant IL1-Ramolecule, is approved for use in treating rheumatoid arthritis andCryopyrin-Associated Periodic Syndromes (CAPS) called Neonatal-OnsetMultisystem Inflammatory Disease (NOMID). It is supplied as a singleuse, glass syringe with 27 gauge needles as a sterile, clear,colorless-to-white, preservative-free solution for daily subcutaneousadministration. Kineret® is formulated in 0.67 ml solution with pH 6.5,containing 100 mg anakinra, 1.29 mg sodium citrate, 5.48 mg sodiumchloride, 0.12 mg disodium EDTA, and 0.70 mg polysorbate 80 in water forinjection. Storage at 2-8° C. is advised. Kineret® is not approved forocular administration.

SUMMARY

Featured herein are stable formulations (e.g., stable aqueousformulations) containing chimeric cytokines (e.g., chimeric cytokines orchimeric cytokine domains as described in WO2012/016203 or in WO2012/103240) that can be used, inter alia, to modulate cellularsignalling responsive to IL-1 family cytokines and their respectivereceptors, to treat disorders, and to detect and/or bind to cellularreceptors, as well as other agents. Described herein is a pharmaceuticalformulation that includes 1 mg/ml to 50 mg/ml of an IL-1β/IL-1Rachimeric cytokine protein. In embodiments, the pharmaceuticalformulation comprises 1 mg/ml to 50 mg/ml of an IL-1β/IL-1Ra chimericcytokine protein; a surfactant; a tonicity agent; and a buffering agent.

In some aspects, the chimeric cytokine protein is P05 or anotherchimeric cytokine such as, e.g., those described in WO2012/016203 or WO2012/103240. In embodiments, the formulation is for topicaladministration. In embodiments, the formulation is for administration tothe eye. In embodiments, the formulation is for topical administrationto the eye. In some embodiments, the formulation has a pH of 5.5 to 7.5,for example a pH of 6.0 to 7.0.

In embodiments, the formulation does not contain a viscosity agent.Certain embodiments relate to a formulation that also contains aviscocity agent. In embodiments, the viscosity agent is a sodiumcarboxymethyl cellulose; an hydroxy ethyl cellulose; an hydroxypropylmethylcellulose; a polyvinyl alcohol; and/or a glycerin. In embodiments,sodium carboxymethyl cellulose is included in the formulation at aconcentration of 0.1-6% w/v.

The formulation typically has a purity of at least 90%, 93%, 95%, or 98%after storage for at least 60 days at 25° C. In some aspects, theformulation has a purity of at least 90%, 93%, 95%, or 98% after storagefor at least four months at 25° C. In other aspects, the formulation hasa purity of at least 90% after storage for a period of at least 2 weeksat 40° C. In some cases, the formulation is stable for at least twoyears when stored at 2° C. to 8° C. In some cases the formulation isstable for at least six months when stored at 25° C. In some cases theformulation is stable for at least eight months when stored at 25° C.

In some embodiments, the surfactant is a non-ionic surfactant, forexample, pluronic acid F-68 (poloxamer 188), polysorbate-20, orpolysorbate-80. In embodiments, the surfactant is pluronic acid F-68(poloxamer 188), and the surfactant is present in a concentration ofabout 0.1% w/v. In some cases, the surfactant is pluronic acid F-68(poloxamer 188), and the surfactant is present in a concentration of0.1% w/v.

The tonicity agent in a formulation can be, for example, sodiumchloride, sorbitol, mannitol, sucrose, or trehalose. In embodiments, thetonicity agent is sorbitol, and the sorbitol is present in aconcentration of about 5% w/v. In certain embodiments, the tonicityagent is sorbitol, and the sorbitol is present in a concentration of 5%w/v.

The buffering agent in the formulation is generally a weak bufferingagent. In embodiments, the buffering agent is a phosphate a citrate, anacetate, a borate, and/or a succinate. The buffering agent can be apharmaceutically acceptable salt of a phosphate, a citrate, an acetate,a borate, or a succinate. In some embodiments, the buffering agent ispresent in an amount of from about 10 mM to about 50 mM. In embodiments,the buffering agent is present at a concentration of 20 mM or less. Inembodiments, the buffering agent, e.g., the sodium citrate and/or sodiumphosphate, is present at a total concentration of 5-15 mM. In someembodiments, the buffering agent, e.g., sodium citrate, is present at aconcentration of 5-15 mM, 7-13 mM, 8-12 mM, or 9-11 mM.

In some cases, the buffering agent is sodium citrate and is present inthe formulation at a concentration of about 10 mM. In some cases, thebuffering agent is sodium citrate and is present in the formulation at aconcentration of 10 mM.

In certain embodiments, the formulation includes an IL-1β/IL-1Rachimeric cytokine protein such as P05; 10 mM sodium citrate; 5% w/vsorbitol; and 0.1% w/v poloxamer 188 (poloxamer F-68), and the pH of theformulation is 6.0.

In some embodiments, the formulation further includes an amino acid. Forexample, the amino acid is arginine, glutamic acid, histidine, ormethionine.

In one example, a chimeric cytokine (e.g., a chimeric cytokinepolypeptide), e.g., a chimeric cytokine containing sequences derivedfrom an Il-1β and an IL-1Ra, is formulated at concentrations of 5 mg/mlto 20 mg/ml (e.g., at a concentration of 1 mg/ml, 5 mg/ml, or 20 mg/ml)in 10 mM sodium citrate, pH 6.0 containing 5% w/v sorbitol and 0.1% w/vpoloxamer, e.g., poloxamer 188 (also referred to as, for example,Lutrol® F-68 (also referred to herein as Lutrol®), Kolliphor® P 188, andpoly(ethylene glycol)-block-poly(ethylene glycol)). In embodiments, thechimeric cytokine is selected from one or more of P01, P02, P03, P04,P05, P06, and P07. In embodiments, the chimeric cytokine is P05.

In embodiments, the components of a formulation described herein arepresent in amounts that may vary around the values provided herein by upto 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%. In embodiments, thecomponents of a formulation are present in amounts that vary around thevalues provided herein by 10%. In embodiments, the formulation comprises9.5-10.5 mM, 9-11 mM, 8.5-11.5 mM, 8-12 mM, 7.5-12.5 mM, 7-13 mM, 6-14mM, or 5-15 mM sodium citrate. In embodiments, the formulation comprises4.75-5.25%, 4.5-5.5%, 4.25-5.75%, 4-6%, 3.75-6.25%, 3.5-6.5%, 3-7%, or2.5-7.5% w/v sorbitol. In embodiments, the formulation comprises0.095-0.105%, 0.09-0.11%, 0.085-0.115%, 0.08-0.12%, 0.075-0.125%,0.07-0.13%, 0.06-0.14%, or 0.05-0.15% w/v poloxamer 188. In embodiments,the concentration of the therapeutic protein (e.g., the chimericcytokine, e.g., P05) in the formulation is 1-50 mg/ml, 1-25 mg/ml, or1-20 mg/ml. In embodiments, the concentration of the therapeutic proteinis 4.75-5.25 mg/ml, 4.5-5.5 mg/ml, 4.25-5.75 mg/ml, 4-6 mg/ml, 3.75-6.25mg/ml, 3.5-6.5 mg/ml, 3-7 mg/ml, or 2.5-7.5 mg/ml. In embodiments, thepH of the formulation is 5.5 to 7.5, or 5.5 to 6.5.

In embodiments, the formulation comprises 8-12 mM sodium citrate, 4-6%w/v sorbitol, 0.08-0.12% w/v poloxamer 188, and 4-6 mg/ml P05. Inembodiments, the pH of the formulation is 5.5 to 7.5. In embodiments,the pH is 5.5 to 6.5. In embodiments, the pH is 6 to 7.

In embodiments, the formulation comprises 9-11 mM sodium citrate,4.5-5.5% w/v sorbitol, 0.09-0.11% w/v poloxamer 188, and 4.5-5.5 mg/mlP05. In embodiments, the pH of the formulation is 5.5 to 7.5. Inembodiments, the pH is 5.5 to 6.5. In embodiments, the pH is 6 to 7.

In embodiments, an IL-1 inhibitor, e.g., anakinra, is formulated at anappropriate concentration (e.g., at a concentration of 5 mg/ml to 100,e.g., 5 to 50 mg/ml, e.g., 5 to 20 mg/me in 10 mM sodium citrate, pH 6.0containing 5% w/v sorbitol and 0.1% w/v poloxamer, e.g., poloxamer 188.In embodiments, the amounts of the components of the formulation mayvary around the values provided herein by up to 5%, 10%, 15%, 20%, 25%,30%, 40%, or 50%. In embodiments, the pH is 5.5 to 7.5. In embodiments,the pH is 5.5 to 6.5. In embodiments, the pH is 6 to 7.

In embodiments, a formulation described herein further comprises aviscosity agent, e.g., sodium carboxymethyl cellulose (CMC). Inembodiments, the formulation comprises CMC, e.g., CMC at a concentrationof 0.1-1% w/v, 0.1-0.5% w/v, or 0.2-0.3 w/v %.

Also provided herein is a method for treating a subject having anIL-1-related disorder. The method includes administering to the subjecta therapeutically effective amount of a composition comprising aformulation described herein. In embodiments, the method includesidentifying a subject having an IL-1-related disorder such as, e.g., adry eye disorder; and administering to the subject a therapeuticallyeffective amount of a composition comprising a formulation as describedherein.

Also described herein is a method of inhibiting IL-1 activity in asubject. The method includes administering to the subject a formulationas described herein. In embodiments, the subject has an IL-1-relateddisorder, e.g., a dry eye disorder.

In some embodiments, the invention relates to a drug delivery devicecomprising a formulation as described herein.

Also disclosed herein is the use of a composition as described herein inthe manufacture of a medicament for treating or preventing anIL-1-related disorder in a subject, e.g., in the manufacture of amedicament for topical administration to a subject for treating orpreventing an IL-1 related disorder in the subject. In embodiments, themedicament is for administration to the eye, e.g., for topicaladministration to the eye. In some embodiments, the medicament is avehicle formulation, e.g., an aqueous formulation comprising orconsisting of sorbitol, sodium citrate, and poloxamer 188 as describedherein. In embodiments, the vehicle formulation is substantially free of(e.g., does not comprise) a therapeutic protein.

In general, the subject treated as described herein is a human or othermammal such as a dog or cat.

In some embodiments, the invention relates to a container or devicecomprising a formulation as described herein. In embodiments, thecontainer is a blow fill seal container.

Applicants have also discovered a pharmaceutical formulation (e.g., avehicle formulation) comprising a surfactant, a tonicity agent, and abuffering agent that can be useful for treating dry eye. In someembodiments, the formulation does not contain a protein or peptide,e.g., the formulation does not contain a therapeutic protein or peptide.In some embodiments, the surfactant is Pluronic F68 (poloxamer 188), thebuffering agent is citrate, and the tonicity agent is sorbitol. In someembodiments, the formulation comprises a chimeric cytokine protein,e.g., a chimeric cytokine protein as described herein or in WO2012/103240, e.g., P05. In some embodiments, the pharmaceuticalformulation also includes a viscocity agent, e.g., CMC. In someembodiments, the pharmaceutical formulation is suitable for use in theeye (i.e., suitable for ocular delivery), e.g., for treating oculardisease such as signs and/or symptoms of dry eye.

Naturally occurring proteins referenced herein specifically includehuman forms of such proteins, and as well as forms from other mammalianspecies.

Embodiments described herein include the following:

Embodiment 1

An aqueous formulation comprising sodium citrate or sodium phosphate ata concentration of 8 to 12 mM; sorbitol at 4% to 6% (w/v); poloxamer 188at a concentration of 0.08% to 0.12% (w/v); and optionally sodiumcarboxymethyl cellulose, wherein the formulation has a pH of 5.5 to 7.5and wherein the formulation is effective for treating an oculardisorder.

Embodiment 2

The formulation of embodiment 1, wherein the formulation has a pH of 5.5to 6.5.

Embodiment 3

The formulation of embodiment 1 or embodiment 2, wherein the formulationis substantially free of a therapeutic protein.

Embodiment 4

The formulation of any one of embodiments 1 to 3, wherein theformulation comprises sodium citrate at a concentration of 8 toll mM;sorbitol at 4.5 to 5.5% (w/v); and poloxamer 188 at a concentration of0.09 to 0.11%.

Embodiment 5

The formulation of embodiment 4, wherein the formulation consists ofsodium citrate at a concentration of 9 toll mM; sorbitol at 4.5 to 5.5%(w/v); and poloxamer 188 at a concentration of 0.09 to 0.11%.

Embodiment 6

The formulation of embodiment 5, wherein the formulation consists ofsodium citrate at a concentration of 9 toll mM; sorbitol at 4.5 to 5.5%(w/v); and poloxamer 188 at a concentration of 0.09 to 0.11%.

Embodiment 7

The formulation of any one of embodiments 1 to 6, comprising sodiumcarboxymethyl cellulose at a concentration of 0.1-1% (w/v).

Embodiment 8

An aqueous formulation comprising sodium citrate at a concentration of9-11 mM; sorbitol at 4.5-5.5% (w/v); and poloxamer 188 at aconcentration of 0.09-0.11%, wherein the formulation has a pH of 5.7 to6.3, wherein the formulation is substantially free of therapeuticprotein, and wherein the formulation is effective for treating an oculardisorder (e.g., an ocular disorder described herein).

Embodiment 9

The formulation any one of embodiments 1 to 8, wherein the oculardisorder is dry eye disease.

Embodiment 10

The formulation of any one of embodiments 1 to 9, wherein theformulation is effective to reduce eye pain or soreness, OSDI score,and/or corneal fluorescein staining (CFS) score.

Embodiment 11

The formulation of embodiment 10, wherein eye pain or soreness isassessed using a visual analog scale or a question from the OSDI.

Embodiment 12

An aqueous formulation comprising 1-50 mg/ml of an IL-1β/IL-1Ra chimericcytokine protein (e.g., P01, P02, P03, P04, P05, P06, or P07); abuffering agent selected from sodium citrate and sodium phosphate;sorbitol, e.g., at a concentration of 3.5-6.5% (w/v); poloxamer 188,e.g., at a concentration of 0.07-0.13% (w/v); and optionally sodiumcarboxymethyl cellulose (CMC), wherein the formulation has a pH of 5.5to 7.5.

Embodiment 13

The formulation of embodiment 12, wherein the chimeric cytokine proteinis P05.

Embodiment 14

The formulation of embodiment 13, wherein the formulation comprises 1-20mg/ml P05.

Embodiment 15

The formulation of embodiment 13, wherein the formulation comprises 3-7mg/ml P05.

Embodiment 16

The formulation of embodiment 13, wherein the formulation comprises 4-6mg/ml P05.

Embodiment 17

The formulation of embodiment 13, comprising sodium citrate and/orsodium phosphate at a total concentration of 5 mM to 15 mM.

Embodiment 18

The formulation of any one of embodiments 12 to 17, wherein sodiumcitrate is present at a concentration of 5 mM to 15 mM.

Embodiment 19

The formulation of embodiment 18, wherein the sodium citrate is presentat a concentration of 8 mM to 12 mM.

Embodiment 20

The formulation of embodiment 18, wherein the sodium citrate is presentat a concentration of 9 mM to 11 mM.

Embodiment 21

The formulation of any one of embodiments 12 to 20, wherein thepoloxamer 188 is present at a concentration of 0.05% to 0.15% w/v.

Embodiment 22

The formulation of embodiment 20, wherein the poloxamer 188 is presentat a concentration of 0.08% to 0.12% w/v.

Embodiment 23

The formulation of embodiment 20, wherein the poloxamer 188 is presentat a concentration of 0.09% to 0.11% w/v.

Embodiment 24

The formulation of any one of embodiments 12 to 23, wherein the sorbitolis present at a concentration of 2.5% to 7.5% w/v.

Embodiment 25

The formulation of embodiment 24, wherein the sorbitol is present at aconcentration of 4% to 6% w/v.

Embodiment 26

The formulation of embodiment 24, wherein the sorbitol is present at aconcentration of 4.5 to 5.5% w/v.

Embodiment 27

An aqueous formulation comprising 1-25 mg/ml P05; sodium citrate orsodium phosphate at a concentration of 8 mM to 12 mM; sorbitol at 4% to6% (w/v); poloxamer 188 at a concentration of 0.08% to 0.12% (w/v); and,optionally, sodium carboxymethyl cellulose, wherein the formulation hasa pH of 5.5 to 7.5.

Embodiment 28

An aqueous formulation comprising or consisting of 1 mg/ml to 25 mg/mlP05; sodium citrate at a concentration of 8-12 mM; sorbitol at 4% to 6%(w/v); and poloxamer 188 at a concentration of 0.08% to 0.12% (w/v),wherein the formulation has a pH of 5.5 to 7.5.

Embodiment 29

An aqueous formulation comprising or consisting of 1 mg/ml to 25 mg/mlP05; sodium citrate at a concentration of 9 mM toll mM; sorbitol at 4.5%to 5.5% (w/v); and poloxamer 188 at a concentration of 0.09% to 0.11%(w/v); wherein the formulation has a pH of 5.7 to 6.3.

Embodiment 30

An aqueous formulation comprising or consisting of 4-6 mg/ml P05; sodiumcitrate at a concentration of 9-11 mM; sorbitol at 4.5-5.5% (w/v); andpoloxamer 188 at a concentration of 0.09-0.11% (w/v); wherein theformulation has a pH of 5.7-6.3.

Embodiment 31

The formulation of any one of embodiments 12 to 30, wherein theformulation has an osmolality of 270-370 mOsm/kg.

Embodiment 32

The formulation of any one of embodiments 1 to 31, wherein theformulation is suitable for administration to the eye.

Embodiment 33

The formulation of embodiment 30, wherein the formulation is suitablefor topical administration to the eye.

Embodiment 34

The formulation of any one of embodiments 1 to 4 and 8 to 33, whereinthe formulation does not comprise a viscosity agent, e.g., does notcomprise CMC.

Embodiment 35

The formulation of any one of embodiments 12 to 34, wherein theformulation further comprises an amino acid, e.g., arginine, glutamicacid, histidine, or methionine.

Embodiment 36

The formulation of any one of embodiments 12 to 34, wherein theformulation further comprises methionine.

Embodiment 37

The formulation of embodiment 36, wherein the methionine is present inthe formulation at a concentration of 1 to 20 mM.

Embodiment 38

The formulation of embodiment 36 or 37, wherein the formulation hasreduced oxidation, compared to a corresponding formulation that does notcomprise methionine, when the formulation is subjected to storage, e.g.,for at least 4 weeks at 25° C.).

Embodiment 39

The formulation of embodiment 38, wherein the formulation has reducedoxidation, compared to a corresponding formulation that does notcomprise methionine, when the formulation is subjected to storage in amultidose container.

Embodiment 40

The formulation of embodiment 38 or 39, wherein oxidation of theformulation is assessed using RP-HPLC.

Embodiment 41

The formulation of any one of embodiments 12 to 40, wherein theformulation has less than or equal to 50 particles per ml for particles≧10 μm and less than or equal to 5 particles per ml for particles ≧25μm, as assessed using a light obscuration particle count test.

Embodiment 42

The formulation of any one of embodiments 12 to 41, wherein theformulation is stable as indicated by the presence of >90% of themonomeric form of the protein relative to aggregated form aftervortexing the protein solution for 4 hours at room temperature, e.g., at25° C.

Embodiment 43

The formulation of embodiment 42, wherein the percentage of themonomeric form of the protein relative to aggregated form is assessedusing SEC-HPLC.

Embodiment 44

The formulation of any one of embodiments 12 to 43, wherein theformulation is stable after storage for at least 5 months at 2-8° C. and60% relative humidity.

Embodiment 45

The formulation of any one of embodiments 12 to 44, wherein theformulation is stable after storage for at least 5 months under ambientconditions, e.g., at room temperature, e.g., at 25° C.

Embodiment 46

The formulation of any one of embodiments 12 to 45, wherein theformulation is stable after storage for at least 4 months at 2-8° C. and60% relative humidity.

Embodiment 47

The formulation of any one of embodiments 12 to 46, wherein theformulation is stable after storage for at least 4 months under ambientconditions, e.g., at room temperature, e.g., at 25° C.

Embodiment 48

The formulation of any one of embodiments 12 to 47, wherein theformulation is stable after storage for at least 3 months at 2° C. to 8°C. and 60% relative humidity.

Embodiment 49

The formulation of any one of embodiments 12 to 48, wherein theformulation is stable after storage for at least 3 months under ambientconditions, e.g., at room temperature, e.g., at 25° C.

Embodiment 50

The formulation of any one of embodiments 12 to 49, wherein theformulation is stable after storage for at least 2 months at 2-8° C. and60% relative humidity.

Embodiment 51

The formulation of any one of embodiments 12 to 50, wherein theformulation is stable after storage for at least 2 months under ambientconditions, e.g., at room temperature, e.g., at 25° C.

Embodiment 52

The formulation of any one of embodiments 12 to 51, wherein theformulation is stable after storage for at least 1 month at 2-8° C. and60% relative humidity.

Embodiment 53

The formulation of any one of embodiments 12 to 52, wherein theformulation is stable after storage for at least 1 month under ambientconditions, e.g., at room temperature, e.g., at 25° C.

Embodiment 54

The formulation of any one of embodiments 12 to 53, wherein theformulation is stable as indicated by the presence of less than or equalto 50 particles per ml for particles ≧10 μm, less than or equal to 5particles per ml for particles ≧25 μm, and less than or equal to 2particles per ml for particles ≧50 μm, e.g., as assessed using amicroscopic particle count test.

Embodiment 55

The formulation of any one of embodiments 12 to 54, wherein theformulation is stable as indicated by the presence of >90% of themonomeric form of the protein relative to aggregated form as assessedusing SEC-HPLC.

Embodiment 56

The formulation of any one of embodiments 12 to 55, wherein theformulation is stable as indicated by conformity of the main band toreference standard in a reduced SDS-PAGE.

Embodiment 57

The formulation of any one of embodiments 12 to 56, wherein theformulation is stable as indicated by conformity of the main band toreference standard in a nonreduced SDS-PAGE.

Embodiment 58

The formulation of any one of embodiments 12 to 57, wherein theformulation is stable as indicated by a main peak of greater than orequal to 85% when the formulation is assessed using weak cation exchangeHPLC (WCEX-HPLC).

Embodiment 59

The formulation of embodiment 58, wherein the formulation comprises P05and is stable as indicated by the presence of less than 10% of thedes-Ala form of P05 as assessed using WCEX-HPLC.

Embodiment 60

The formulation of any one of embodiments 12 to 59, wherein theformulation is packaged in a blow fill seal container.

Embodiment 61

The formulation of any one of embodiments 44 to 53, wherein said storageis storage in a blow fill seal container.

Embodiment 62. A method of treatment, the method comprisingadministering to a subject having an IL-1-related disorder a formulationaccording to any one of embodiments 1 to 61, thereby treating the IL-1related disorder Embodiment 63

The method of embodiment 62, wherein the IL-1 related disorder is a dryeye disorder.

Embodiment 64

A method of treating an ocular disorder, e.g., a dry eye disorder, themethod comprising administering to a subject having the ocular disorder,e.g., the dry eye disorder, an aqueous formulation comprising sodiumcitrate or sodium phosphate at a concentration of 8 mM to 12 mM;sorbitol at 4% to 6% (w/v); poloxamer 188 at a concentration of 0.08% to0.12% (w/v); and optionally sodium carboxymethyl cellulose; wherein theformulation has a pH of 5.5 to 7.5 and is substantially free oftherapeutic protein, thereby treating the dry eye disorder.

Embodiment 65

The method of embodiment 64, wherein the aqueous formulation comprisessodium citrate at a concentration of 8 mM to 11 mM, sorbitol at 4.5% to−5.5% (w/v) and poloxamer 188 at a concentration of 0.09% to 0.11%.

Embodiment 66

The method of embodiment 64, wherein the aqueous formulation consists ofsodium citrate at a concentration of 8-11 mM, sorbitol at 4.5-5.5% (w/v)and poloxamer 188 at a concentration of 0.09-0.11%.

Embodiment 67

A method of treating a dry eye disorder, the method comprisingadministering to a subject having a dry eye disorder an aqueousformulation comprising 1 to 25 mg/ml P05; sodium citrate or sodiumphosphate at a concentration of 8 mM to 12 mM; sorbitol at 4% to 6%(w/v); poloxamer 188 at a concentration of 0.08% to 0.12% (w/v); andoptionally sodium carboxymethyl cellulose, wherein the formulation has apH of 5.5 to 7.5, thereby treating the dry eye disorder.

Embodiment 68

A method of treating a dry eye disorder, the method comprisingadministering to a subject having a dry eye disorder an aqueousformulation consisting of 1 mg/ml to 25 mg/ml P05; sodium citrate at aconcentration of 8 mM to 12 mM; sorbitol at 4% to 6% (w/v); poloxamer188 at a concentration of 0.08% to 0.12% (w/v), wherein the formulationhas a pH of 5.5 to 7.5, thereby treating the dry eye disorder.

Embodiment 69

A method of treating a dry eye disorder, the method comprisingadministering to a subject having a dry eye disorder an aqueousformulation comprising or consisting of 1 mg/ml to 25 mg/ml P05; sodiumcitrate at a concentration of 9 mM to 11 mM; sorbitol at 4.5% to 5.5%(w/v); and poloxamer 188 at a concentration of 0.09% to 0.11% (w/v),wherein the formulation has a pH of 5.7 to 6.3, thereby treating the dryeye disorder.

Embodiment 70

The method of any one of embodiments 62 to 69, wherein the method iseffective to reduce eye pain or soreness, OSDI score, and/or cornealfluorescein staining (CFS) score.

Embodiment 71

The method of embodiment 70, wherein eye pain or soreness is assessedusing a visual analog scale or a question from the OSDI.

Embodiment 72

The method of any one of embodiments 62 to 70, wherein the formulationis administered one to five times per day.

Embodiment 73

The method of any one of embodiments 62 to 72, wherein the formulationis administered topically.

Embodiment 74

The method of embodiment 73, wherein the formulation is administeredtopically to the eye.

Embodiment 75

The method of any one of embodiments 62 to 74, wherein the formulationis administered three times per day.

Embodiment 76

The method of any one of embodiments 62 to 71 or 73 to 74, wherein theformulation is administered ad libitum.

Embodiment 77

A container or device comprising the formulation of any one ofembodiments 1 to 61.

Embodiment 78

The container or device of embodiment 77, wherein the container ordevice has been stored at 25° C. for at least two weeks, e.g., for atleast four weeks, and is substantially free of particulates.

Embodiment 79

A blow fill seal container comprising the formulation of any one ofembodiments 1 to 61.

Embodiment 80

A multidose container comprising the formulation of any one ofembodiments 1 to 61.

Embodiment 81

A multidose container comprising the formulation of embodiments 35 to40.

Embodiment 82

A drug delivery device comprising a formulation of any one ofembodiments 1 to 61.

Embodiment 83

The drug delivery device of embodiment 82, wherein the drug deliverydevice is a blow fill seal container.

Embodiment 84

The container or device of any one of embodiments 77 to 83, wherein thecontainer or device is sealed in a pouch, optionally containing an inertgas, e.g., nitrogen or argon.

Embodiment 85

The formulation of any one of embodiments 1 to 61, for use in treatingan IL-1 related disorder, e.g., a dry eye disorder.

Embodiment 86

Use of a formulation of any one of embodiments 1 to 61 in themanufacture of a medicament for treating an IL-1-related disorder in asubject.

Embodiment 87

A kit comprising a container or device comprising the formulation of anyone of embodiments 1 to 61, and optionally, instructions for use.

The foregoing embodiments are not necessarily separate embodiments. Insome cases, they may be combined with each other and/or with otheraspects and embodiments disclosed herein.

All patents, published patent applications, and published referencescited herein are incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reproduction of an SDS-PAGE gel showing exemplary samples ofprotein purified from E. coli expressing receptor binding agents. The 15and 20 kDa molecular weight markers are indicated at left. Lanes are asfollows: molecular weight marker (lanes 1 and 6), extract (lanes 2 and7), material purified by cation exchange chromatography (lanes 3 and 8),material additionally purified by anion exchange chromatography (lanes 4and 9), and reduced samples of such material (lanes 5 and 10). Lanes 2-5are of P05 purification, and Lanes 6-10 are of P04 purification. Seealso Example 2.

FIG. 2A is a table and accompanying bar graph illustrating the resultsof an experiment testing the ability of the P06, P07, and P01 proteinsto agonize signaling relative to IL-1β and a negative control,[3-glucuronidase (GUS) protein.

FIG. 2B is a graph depicting the results of an experiment testing theability of P01 to antagonize IL-1β activity at various IL-1βconcentrations.

FIG. 3A is a graph depicting the results of an experiment testingantagonism of IL-1β by P03 (hexa-histidine tagged (SEQ ID NO: 23)), P04(hexa-histidine tagged (SEQ ID NO: 23)), P05 (hexa-histidine tagged (SEQID NO: 23)), and IL-1Ra in the presence of 0.1 ng/ml IL-1β (human).

FIG. 3B is a graph depicting the results of an experiment testingantagonism of IL-1β by lysates containing untagged forms of P01, P02,P03, P04, and P05, and IL-1Ra in the presence of 0.1 ng/ml IL-1β (human)and using estimates of the concentration of protein in the respectivelysates.

FIG. 4A is a graph showing the dynamic light scattering (DLS) resultsfor P05 in a phosphate formulation (P05 at 20 mg/ml, 10 mM phosphate, 5%w/v sorbitol, 0.1% w/v poloxamer 188, pH 6.5).

FIG. 4B is a graph showing DLS results for P05 in a citrate formulation(P05 at 20 mg/ml, 10 mM citrate, 5% w/v sorbitol, 0.1% w/v poloxamer188, pH 6.0).

FIG. 5A is a graph depicting thermal denaturation of IL-1Ra, IL-1β, P03,P04, and P05 as described in Example 8.

FIG. 5B is a graph depicting the negative first derivative of the graphin FIG. 5A (the negative first derivative provides improvedvisualization of the melting temperature).

FIG. 6A is a bar graph depicting the mean corneal staining score±SEM astested by fluorescein staining of the cornea per eye of two independentstudies, on days 0, 3, 7, 9, and 11 for mice in a dry eye model. Themice received no treatment (n=18), 10 mg/ml P05 (n=19), or 1.25×PBS, thevehicle (n=20). Asterisks indicate statistical significance of P05relative to vehicle as follows: * (P<0.05) and ** (P<0.005).

FIG. 6B is a bar graph representing data showing mean corneal stainingscore±SEM of the cornea per eye, on days 0, 3, 7, 9, and 11 for mice ina dry eye model. The mice received no treatment (n=8), 1.25×PBS vehicle(n=8), 10 mg/ml murine serum albumin (MSA) (n=8), or 10 mg/ml P05 (n=9).Asterisks indicate statistical significance of P05 relative to murineserum albumin as follows: * (P<0.05) and *** (P<0.0005).

FIG. 6C is a bar graph representing data for mice that were treated withRestasis® (0.05% cyclosporine emulsion) (n=8) in the same experiment asFIG. 6B. Asterisks indicate statistical significance of P05 relative tocyclosporine (Restasis®) as follows: ** (P<0.005) and *** (P<0.0005).

FIG. 7 depicts the design of the clinical trial described in Example 16.

FIG. 8 is a graph showing the mean change from baseline in the OSDIscore for the groups of subjects who received EBI-005 formulations(combined data for the groups that received 5 mg/ml and 20 mg/mltreatments) and vehicle formulation.

FIG. 9 is a graph showing the mean change from baseline in pain for thegroups of subjects who received EBI-005 formulations (combined data forthe groups that received 5 mg/ml and 20 mg/ml treatments) and vehicleformulation.

FIG. 10 is a graph showing the mean change from baseline in cornealfluorescein staining (CFS) score for the groups of subjects who receivedEBI-005 formulations (combined data for the groups that received 5 mg/mland 20 mg/ml treatments) and vehicle formulation.

DETAILED DESCRIPTION

Applicants have achieved formulations that are useful for providing aprotein, e.g., a chimeric cytokine polypeptide such as an IL-1β/IL-1Rachimera to a subject in need of treatment with such a formulation. Theformulations are generally useful for formulation of proteincompositions requiring stability, e.g., proteins that are susceptible toagitation, are susceptible to oxidation, e.g., due to methionineresidues, or susceptible to deamindation, e.g., due to asparagine orarginine residues. Also disclosed herein are methods of preparing andadministering such formulations.

In some embodiments, a formulation comprises a chimeric cytokinepolypeptide, e.g., a chimeric polypeptide containing selected sequencesderived from an IL-1β and an IL-1Ra sequence, that are suitable forpharmaceutical use, for example, for ophthalmic use, including effectivetopical treatment for an IL-1-related disorder. In general, formulationsdescribed herein are surprisingly stable, even at relatively highconcentrations of the polypeptide, e.g., at concentrations suitable forstorage of bulk drug substance as well as at concentrations suitable forfor treating a subject. An advantage of this feature is that it is notnecessary to remove undesirable agents from the bulk drug substance inorder to formulate the drug for patient use.

Notably, Applicants have successfully achieved an effective aqueousformulation of chimeric cytokine polypeptides that is suitable fortopical administration, e.g., in the eye, e.g., to the front or cornealsurface of the eye. To the best of applicants' knowledge, as of thisfiling there are no approved biologic drugs (biologics) for topicalophthalmic administration approved by the FDA. Furthermore, applicantswere able to formulate such a polypeptide at a pH that is compatiblewith administration to the eye (e.g., a pH of 4.5 to 7.0, 5.5 to 7.0,5.5 to 6.5, or 6.0 to 7.0), and contains a components that render theformulation comfortable for subjects being treated with the polypeptide.Patients are more likely to be compliant with treatment if theformulation is comfortable, e.g., does not cause irritation. Inembodiments, the formulation does not cause one or more symptoms ofirritation such as, e.g., eye redness, tearing, mucous discharge, orsubjective discomfort.

Stability

Topical ophthalmic drugs are generally self-administered by patients.Because the patient may be storing a drug for a relatively long periodof time, the formulation may be subjected to higher temperatures andgreater levels of agitation stress than a formulation that is typicallystored only by a physician or pharmacist prior to administration. As isknown in the art, proteins are more sensitive to agitation andtemperature than small molecules. Agitation stress can lead toprecipitation and heat stress can lead to precipitation and to chemicaldegradation. In addition, during loading of a compound into a deliverydevice, there can be exposure to heat stress. Applicants have achieved aformulation that successfully provides excellent stability when exposedto agitation stress and heat.

Some manufacturing processes require at least brief exposure of aformulation to relatively high temperature. For example, loading aformulation into a blow fill seal (BFS) container can result in exposureof the formulation to elevated temperatures, in addition to agitationassociated with the filling process. Applicants have loaded aformulation into such a device (a BFS container) and demonstratedstability of the formulation immediately following loading and over anextended period of time. In some embodiments, a formulation as providedherein is suitable for use with BFS. In embodiments, a formulation thatis suitable for use with BFS shows stability immediately followingloading into a BFS container and/or after storage in a BFS container,e.g., after storage for periods of time and under conditions describedherein.

In embodiments, a formulation described herein is stable. Inembodiments, the formulation exhibits stability under conditions (e.g.,storage at particular temperatures, or agitation stress) describedherein. In embodiments, stability is assessed using one or more methodsdescribed herein (e.g., based on visual appearance, content byspectrophotometry (A280), SDS-PAGE non-reduced, SDS-PAGE reduced; sizeexclusion HPLC (SE HPLC); reverse phase HPLC (RP-HPLC); weak cationexchange HPLC (WCEX-HPLC); potency; a light obscuration particle counttest (e.g., a light obscuration particle count test as described in USP<788>); or a microscopic particle count test (e.g., a microscopicparticle count test as described in USP <788>)) and/or methods known inthe art.

Stability can be assessed based on visual appearance. In embodiments, aformulation is stable if it is a clear to slightly opalescent colorlesssolution essentially free from visible particulates.

In embodiments, the formulation is stable at about 25° C. to about 40°C., for example, about 27° C., about 28° C., about 29° C., about 30° C.,about 31° C., about 32° C., about 33° C., about 34° C., about 35° C.,about 36° C., about 37° C., about 38° C., about 39° C., or about 40° C.for a period of at least two days; three days; five days; one week; tendays, two weeks, three weeks, four weeks, five weeks, six weeks, eightweeks, 16 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45weeks, one month, two months, three months, four months, five months,six months, seven months, eight months, or more.

In embodiments, the formulations are stable for long periods of timeduring storage at temperatures of from about 2° C. to about 8° C., suchas at about 4° C., about 5° C., about 6° C., from 2° C. to 8° C., at 4°C., at 5° C., or at 6° C. For example, the formulations are stable atsuch storage temperatures for a period of at least two weeks; fourweeks; six weeks; two months; three months; six months, one year, twoyears, three years, or four years.

Stability of a formulation can be assessed, e.g., after storage for atleast 2, 4, 6, 8, 12, or 18 months, e.g., at 2-8° C., or after storageunder ambient conditions, e.g., at room temperature (RT), e.g. at about25° C. for, e.g., at least 2 weeks, 1 month, 2 months, 3 months 5months, 6 months, 12 months, or 18 months. In embodiments, theformulation is stable after storage at 2-8° C. for at least 8 months. Inembodiments, the formulation is stable after exposure to roomtemperature for at least 5 months. In some such embodiments, theformulation is stable after storage, e.g., for at least 5 months, in aBFS container.

Stability can be assessed, e.g., based on methods and criteria describedherein or known in the art. For example, stability can be assessed basedon physical purity (e.g., lack of aggregation, e.g., as assessed usingsize exclusion HPLC, also referred to herein as size exclusion, SE HPLC,or SEC HPLC), chemical purity (e.g., as assessed using weak cationexchange HPLC, reverse phase HPLC, and/or SDS PAGE (e.g., reduced ornonreduced SDS PAGE)), and/or the levels of particulates (e.g., asassessed visually or by particle count using an HIAC liquid particlecounter (Beckman Coulter, Brea, Calif.)).

In embodiments, stability is demonstrated based on compliance withguidelines for particulate matter in opthalmic solutions, e.g., as setforth in USP <789> (U.S. Pharmacopeia, Particulate Matter in OpthalmicSolutions).

In embodiments, the formulation has less than or equal to 50 particlesper ml for particles ≧10 μm and/or less than or equal to 5 particles perml for particles ≧25 μm, e.g., as assessed using a light obscurationparticle count test (e.g., a light obscuration particle count test asdescribed in USP <788>).

In embodiments, the formulation has less than or equal to 50 particlesper ml for particles ≧10 μm, less than or equal to 5 particles per mlfor particles ≧25 μm, and/or less than or equal to 2 particles per mlfor particles ≧50 μm, e.g., as assessed using a microscopic particlecount test (e.g., a microscopic particle count test as described in USP<788>).

In embodiments, stability is demonstrated based on compliance withguidelines for particulate matter in injections, e.g., as set forth inUSP <788> (U.S. Pharmacopeia, Particulate Matter in Injections).

In embodiments, the formulation has less than or equal to 6000 particlesper container (for containers with a volume of 100 ml or less) forparticles ≧10 μm, and/or less than or equal to 600 particles percontainer (for containers with a volume of 100 ml or lower) forparticles ≧25 um, e.g., as assessed using a light obscuration particlecount test (e.g., a light obscuration particle count test as describedin USP <788>).

In embodiments, the formulation has less than or equal to 3000 particlesper 5 ml for particles ≧10 m and/or less than or equal to 300 particlesper 5 ml for particles ≧25 um, e.g., as assessed using a microscopicparticle count test (e.g., a microscopic particle count test asdescribed in USP <788>).

In embodiments, the protein in a formulation is protected from agitationstress as demonstrated, e.g., by lack of aggregation (lack ofaggregation may be demonstrated, e.g., if the formulation containscontains >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%of the monomeric form of the protein relative to aggregated form) aftervortexing the protein solution, e.g., for 1-8 hours at room temperature(RT), e.g., for 4 hours at RT. Aggregation can be assessed, e.g., usingmethods described herein or methods known in the art. For example,aggregation can be assessed using ultracentrifugation, size-exclusionchromatography, gel electrophoresis, dynamic light scattering, and/orturbidity measurements.

In some aspects, stability is assayed by physical or chemical methodsknown in the art. For example, physical purity or lack of aggregationcan be determined using size exclusion HPLC or other methods thatdetermine the relative amount of monomeric polypeptide in a formulation.Typically, a formulation with acceptable stability contains >90% of themonomeric form of therapeutic protein (e.g., the chimeric cytokine,e.g., P05) relative to aggregated forms of the protein. In embodiments,the formulation contains >90%(e.g., >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) of themonomeric form of the therapeutic protein (e.g., the chimeric cytokine,e.g., P05), relative to aggregated forms of the protein.

Chemical purity can be determined, for example, using weak cationexchange HPLC or reverse phase HPLC. Typically, a formulation withacceptable stability contains >80% of the native molecule, relative tochemically modified forms of the molecule, e.g., as assessed using weakcation exchange HPLC. In embodiments, the formulation contains >80%(e.g., >85%, >87%, >90%, or >95%) of the native molecule, relative tochemically modified forms of the molecule (e.g., oxidized or acetylatedforms).

Particulates may be identified visually. In embodiments, the formulationis one that is essentially free of particulates that can be identifiedvisually.

Applicants note that information on anakinra, an IL-1Ra, formulated fordelivery by injection states that the product has a shelf life of threeyears, is to be stored at 3-8° C., and “For the purpose of ambulatoryuse, Kineret® may be removed from the refrigerator for 12 hours attemperature not above 25° C., without exceeding the expiry date. At theend of this period, the product must not be put back in the refrigeratorand must be disposed of.” (See:medicines.org.uk/EMC/medicine/23104/SPC/Kineret+100+mg+solution+for+injection+in+a+pre-filled+syringe#SHELF_LIFE).This provides a contrast to the surprising stability of, for example,the P05 formulation provided herein.

Biologic treatments can be problematic to administer because they canhave a relatively short shelf life or require special storage conditionsthat can create obstacles for storage, transport, and patient use aswell as assuring a sufficient supply of the biologic. An advantage ofcertain formulations provided herein is that the formulations aresurprisingly stable not only under conditions of refrigeration, but alsoat temperatures that are in accord with room temperature (e.g., 25° C.)and above (e.g., 40° C.). Accordingly, the cytokine protein orpolypeptide formulations (e.g. heterologous cytokine protein orpolypeptide formulations), e.g., formulations described herein are, insome embodiments, provided in a liquid form that is stable at RT (e.g.,at 25° C.) for a period of at least three days, five days, one week, tendays, two weeks, three weeks, six weeks, eight weeks, 16 weeks, 20weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, one month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, twelve months, or more. In embodiments, a month isdetermined on date to date basis, e.g., from the first of the month tothe first of the second month.

In other aspects the formulations are stable at about 25° C. to about40° C., for example, about 27° C., about 28° C., about 29° C., about 30°C., about 31° C., about 32° C., about 33° C., about 34° C., about 35°C., about 36° C., about 37° C., about 38° C., about 39° C., or about 40°C. for a period of at least two days; three days; five days; one week;ten days, two weeks, three weeks, four weeks, five weeks, six weeks,eight weeks, 16 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks,45 weeks, one month, two months, three months, four months, five months,six months, seven months, eight months, or more.

In one example, a formulation is stable for one month at 25° C. and 1week at 40° C. when the protein component of the formulation, e.g., P05,is at a concentration of 20 mg/ml. In another specific embodiment, theformulation, loaded into a blow fill seal vial or blow fill deliverydevice, is stable at 25° C. for at least three months for a formulationcomprising protein, e.g., P05, at a concentration of 1 mg/ml, 5 mg/ml,or 10 mg/ml. In some embodiments, this formulation is stable for atleast eight months.

In embodiments, a formulation comprising 4.5-5.5 mg/ml P05, 9-11 mMsodium citrate; 4.5-5.5% w/v sorbitol, and 0.09-0.11% w/v poloxamer 188is stable for at least five months at 2° C. to 8° C. and/or at roomtemperature, e.g., at 25° C. In some embodiments, a formulationconsisting of 10 mM Na citrate, pH 6.0, 5% sorbitol, 0.1% poloxamer, and5 mg/ml or 20 mg/ml P05 is stable for at least five months at 2° C. to8° C. and/or at room temperature, e.g., at 25° C. for at least 5 months.

Concentration

A further problem in administering biologics, is providing a sufficientconcentration of the biologic. This is a particular problem inophthalmic applications in which it is desirable to provide a relativelyhigh concentration of the biologic so as to achieve a therapeutic effectwith a minimum number of doses. Applicants have been able to achieve aformulation that can deliver an effective dose of a chimeric cytokineformulation containing a high concentration, or a therapeuticallyeffective concentration, of the polypeptide that does not appreciablyaggregate, precipitate, or lose chemical purity when stored underconditions such as those described supra and elsewhere within thisspecification. Furthermore, applicants have demonstrated stability of acytokine formulation at protein concentrations of up to 80 mg/ml, e.g.,50 mg/ml in a formulation comprising a tonicity agent, a surfactant, anda buffering agent. Therefore, in one aspect, a formulation featured inthe invention contains a chimeric cytokine polypeptide stably present inthe formulation in a concentration of from 0.1 mg/ml to 100 mg/ml,0.1-80 mg/ml, 0.1 to 50 mg/ml, 0.1 mg/ml to 20 mg/ml, 0.1 mg/ml to 5mg/ml, 0.1 mg/ml to 1 mg/ml, 1 mg/ml to 100 mg/ml; 5 mg/ml to 100 mg/ml;5 mg/ml to 30 mg/ml; 10 mg/ml to 100 mg/ml; 10 mg/ml to 30 mg/ml; 20mg/ml to 100 mg/ml; 30 mg/ml to 100 mg/ml; 40 mg/ml to 100 mg/ml; 50mg/ml to 100 mg/ml; 60 mg/ml to 100 mg/ml; 1 mg/ml to 80 mg/ml; 5 mg/mlto 80 mg/ml; 10 mg/ml to 80 mg/ml; 20 mg/ml to 80 mg/ml; 40 mg/ml to 80mg/ml; 50 mg/ml to 80 mg/ml; 60 mg/ml to 80 mg/ml; 1 mg/ml to 60 mg/ml;5 mg/ml to 60 mg/ml; 10 mg/ml to 60 mg/ml; 20 mg/ml to 60 mg/ml; 30mg/ml to 60 mg/ml; 40 mg/ml to 60 mg/ml; or 50 mg/l to 60 mg/ml. Forexample, the formulation contains 0.1 mg/ml, 1 mg/ml, 2 mg/ml, 5 mg/mlto 20 mg/ml, e.g., 5 mg/ml or 20 mg/ml.

Viscocity agents are frequently used in formulations, e.g., forophthalmic use. Such agents are generally included to increase theresidence time of an ophthalmic treatment that would otherwise berapidly cleared by blinking and drainage through the conjunctival sac.However, such agents can have deleterious effects, e.g., allergicsymptoms, damage protein components of a formulation, or cause blurryvision. While such agents can be used in certain formulations describedherein, in some embodiments Applicants have achieved formulations thatdo not require a viscocity agent for the active component, i.e., achimeric cytokine, to be used as an effective therapeutic.

In another aspect the formulations featured in the invention contain oneor more surfactants. Although the use of a surfactant can be useful,e.g., for reducing adhesion of a molecule to a container, reducingaggregation of a protein particularly under conditions of agitation,addition of a surfactant can also render a therapeutic agent unusablebecause of foaming, disruption of natural membranes and other barriers,and unacceptable discomfort caused by treatment. Applicants havesucceeded in providing a formulation that includes a surfactant, butdoes not incur such disadvantages. Typically, the surfactant is anon-ionic surfactant. Surfactants suitable for use in the disclosedformulations can include, but are not limited to: poloxamers, such aspoloxamer 188. In some embodiments, a surfactant is a polysorbate, suchas polysorbate-20 and polysorbate-80. Other surfactants that can beuseful include Cremophor® EL, tyloxapol, octoxynol 40 (Triton® X405, andpolyoxyl 40 stearate. In certain embodiments, a formulation contains asurfactant (e.g., poloxamer 188) in a concentration of about 0.05%,0.06%, 0.1% to 1.0%, 0.1% to 0.5%, 0.2% to 0.5%, or 0.1% to 0.2% w/v,for example, 0.1% w/v poloxamer 188. Suitable surfactants andconcentrations of such surfactants can be determined by testing whetherthe surfactant prevents aggregation in agitation studies. Methods ofconducting such studies are known in the art. For example, it can bedetermined whether surfactant is needed to prevent precipitation fromagitation stress. In such experiments, typically, a screen is performedusing agitation and analysis. Examples of concentrations used for suchstudies are 0.01%, 0.02%, 0.06%, and 0.1% w/v surfactant, e.g.,poloxamer 188. In embodiments, aggregation and/or precipitation areassessed using analysis by spectrophotometry (A₂₈₀), visual inspection,size exclusion chromatography (SEC), light obscuration (e.g., using aHIAC device), or Micro-Flow Imaging™ (MFI, ProteinSimple, Santa Clara,Calif.). A surfactant is generally selected for use in a formulationthat is associated with the least amount of precipitation, e.g., novisible precipitation, or particle count that meets guidelines forparticulate matter in injections (see, e.g., USP <788>) or guidelinesfor particulate matter in ophthalmic solutions (see, e.g., USP <789>).

In another aspect, the formulations featured in the invention containone or more tonicity agents. Suitable tonicity agents include, but arenot limited to: sodium chloride, sorbitol; mannitol, sucrose, trehalose,or other sugars. Without committing to any theory, such agents maycontribute to the surprising stability of a chimeric cytokinepolypeptide. In embodiments, a tonicity agent, e.g., a sugar such as,e.g., sorbitol, provides or contributes to thermal stability. In certainembodiments, the formulations featured in the invention are isotonic forthe eye (e.g., having an osmolality of about 270-330 mOsm per kg). Insome embodiments, the formulation has an osmolality of from about 250 toabout 450 mOsm per kg, 300 to 400 mOsm per kg, 350 to 400 mOsm per kg,200 to 375 mOsm per kg, or 350 to 375 mOsm per kg. In embodiments, theformulation has an osmolality of 270-330 mOsm per kg, e.g., about 320mOsm per kg. Depending upon the tonicity agent, certain embodimentsfeatured in the invention contain from about 1% to about 15% w/v; 2% to12% w/v; 5% to 12% w/v; or 5% to 10% w/v. For sorbitol or mannitol, anexample of a concentration is about 5% w/v, e.g., the concentration is5% w/v. For sucrose or trehalose, an example of a concentration is about9% w/v.

Buffering Agents

In another aspect, a formulation featured herein contains one or morebuffering agents. Suitable buffering agents include, but are not limitedto, phosphates; citrates; acetates; borates; succinates; and TRIS. Insome cases a salt of the buffering agents is a sodium salt or apotassium salt. In certain embodiments featured in the invention, thebuffering agent is present in an amount of from about 10 mM to about 50mM, for example from about 20 mM to about 40 mM, to provide a weakbuffering effect. This allows the formulation to be quickly neutralizedat the administration site, e.g., on the surface of the eye, in theevent of stinging or discomfort. In some embodiments, the bufferingagent is present in an amount of about 10 mM, about 15 mM, about 20 mM,about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM or about50 mM. In some formulations, the buffering agent is a citrate, e.g.,sodium citrate. In other formulations, the buffering agent is citratepresent at 10 mM. In general, the buffering agent is a weak bufferingagent.

In general, a suitable buffer is selected by conducting a stabilitystudy in which the polypeptide of interest is exposed to various buffersat various pH's, concentrations, temperatures, and for various times.Buffers can be selected, for example by placing the polypeptide ofinterest in the buffer and subjecting the samples to elevatedtemperatures (accelerated stability testing) then test for physicalstability (precipitation by visual inspection) or chemical stability,for example, by monitoring deamidation by weak cation exchangechromatography or oxidation by reversed phase chromatography. Additionalassays can include monitoring of A₂₈₀, SDS-PAGE, pH, and osmolality. Abuffer that provides the best physical and chemical stability isselected.

Amino Acids

In another aspect, a formulation featured in the invention contains oneor more amino acids. Suitable amino acids include, but are not limitedto: arginine, glutamic acid, histidine, or methionine. The amino acid istypically selected to enhance the stability and/or the solubility of theprotein. Methods of identifying such amino acids are known in the art.In some embodiments, a formulation such as a P05 formulation containshistidine or methionine.

In some embodiments a formulation contains an oxygen scavenger, e.g.methionine. In some embodiments, the formulation is in a plasticcontainer. In embodiments, the plastic container is sterilized using amethod that generates free radicals, e.g., the container is sterilizedusing gamma radiation or ethylene oxide. In some such embodiments, theformulation includes methionine, e.g., methionine at a concentration of1-20 mM. In embodiments, methionine is present at a concentration of 1-5mM, 5-10 mM, 10-15 mM, 15-20 mM, or 5-15 mM. In embodiments, methionineis present at about 1 mM, 5 mM, 10 mM, 15, mM, or 20 mM. In embodiments,the formulation comprises methionine at a concentration of about 5 mM,e.g., at a concentration of 2.5-7.5 mM, 3-7 mM, or 4-6 mM. In someembodiments a formulation comprising methionine in a sterilized plasticcontainer and the amount of oxidation is less than that of acorresponding formulation that does not contain methionine.

Viscocity Agents

In another aspect, formulations featured in the invention may containone or more viscosity agents. Suitable viscosity agents include, but arenot limited to, methylcelluloses, including sodium carboxymethylcellulose (also referred to herein as carboxymethyl cellulose or CMC);hydroxy celluloses, including ethyl cellulose; hydroxypropylmethylcellulose (hypromellose); carbomers, such as 934P, 971P and 974P;polyvinyl alcohol; xanthan gum; guar gum; gellan gum; and glycerin.

The formulations featured in the invention may also contain otherpharmaceutically acceptable excipients. See e.g., Gennaro (ed.),Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott,Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed.,Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727);Kibbe (ed.), Handbook of Pharmaceutical Excipients, 3rd ed. (2000)(ISBN: 091733096X); Protein Formulation and Delivery, McNally andHastedt (eds.), Informa Health Care (ISBN: 0849379490) (2007). Among theexcipients that can be added are preservatives, penetration enhancersand bioadhesives. Penetration enhancers and bioadhesives may include,for example, chitosan, cytochalasin B, aminated gelatin,poly-ε-caprolectone (carbopol 941P); poly(butylcyanoacrylate);poly-L-arginine; cyclodextrins; gellan; poly(acrylic acid); hyaluronicacid; mucin; alginate; a carbophil, and poloxamers (e.g., see Nagarwalet al., J Controlled Release, 136:2-13 (2009); Ding, PSTT 1:328-35(1998); and Sahoo et al., Drug Discovery Today, 13:144-51(2008). Otherexcipients may be useful as stabilizers, and can include, for example,glycerin, potassium chloride, potassium phosphate, propylene glycol,sodium acetate, sodium bisulfite, sodium borate, sodium boratedecahydrate, sodium chloride, sodium citrate, sodium phosphate, sodiumphosphate (including sodium phosphate monobasic and dibasic); zincchloride, phenol, benzoate, derivatives of castor oil and ethyleneoxides, and Cremophor® (BASF Corp., Germany) Pharmaceutical compositionsfeatured in the invention can be formulated in a variety of forms.

These include, for example, liquid, semi-solid, and solid dosage forms,such as liquid solutions (e.g., injectable and infusible solutions),dispersions or suspensions, including nanoparticles and liposomes. Theform will generally depend on the intended mode of administration andtherapeutic application. Compositions for the agents described hereinare typically in the form of injectable or infusible solutions, or areformulated for topical delivery, e.g., topical ocular delivery.

In some embodiments, a pharmaceutical composition described herein issterile and stable under the conditions of manufacture and storage. Apharmaceutical composition can also be tested to ensure it meetsregulatory and industry standards for administration. The compositioncan be formulated as a solution, microemulsion, dispersion, liposome, orother ordered structure suitable to high drug (e.g., a biologic)concentration. Sterile injectable solutions can be prepared byincorporating an agent described herein in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating an agent described herein intoa sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,exemplary methods of preparation include vacuum drying and freeze-dryingthat yields a powder of an agent described herein plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be engineered byinclusion of an agent that delays absorption, for example, monostearatesalts and gelatin. Such an agent may be particularly useful in alow-dose formulation. In embodiment, the formulation comprises ≦1 mg/mlof a therapeutic protein (e.g., a chimeric cytokine, e.g., P05) andgelatin is included in the formulation.

In certain embodiments, a formulation is prepared with a carrier, e.g.,to extend the pharmacokinetics (PK) of a chimeric cytokine polypeptide(e.g., as assessed based on its half-life in the body, e.g., in the eye,e.g., on the cornea). In such embodiments, the chimeric cytokinepolypeptide can be delivered, for example, as a controlled releaseformulation, delivered by an implant or a microencapsulated deliverysystem. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. See e.g., Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,New York, 1978.

A feature of formulations described herein is that they do not contain apreservative. In general, preservatives can affect the chimeric cytokinepolypeptide, e.g., causing changes to the structure of the polypeptide.In addition, preservatives can cause in a subject, for example, aninflammatory response, which is antithetical to the desired treatmenteffect. Formulations are sterile, stored, and filled into their finalcontainers under sterile conditions.

Vehicle Formulations

Applicants have also unexpectedly discovered that a formulation asdescribed herein that does not contain a therapeutic protein (e.g., avehicle only formulation) is useful for treating one or more signs orsymptoms of ocular disease, e.g., dry eye disease, e.g., signs orsymptoms of dry eye disease described herein. In some embodiments, avehicle formulation comprises a surfactant, a tonicity agent, and abuffering agent. In some such embodiments, the formulation is effectiveto decrease pain (e.g., pain as assessed using the pain question scoreor a visual analog scale), the OSDI, or a subscale of the OSDI. In somesuch embodiments, the formulation decreases corneal fluorescein staining(CFS). As used herein, the term “vehicle only formulation” specificallyrefers to a formulation described herein that is substantially free ofprotein or peptide components, e.g., does not contain a therapeuticprotein. It is to be understood that a vehicle formulation describedherein can contain any therapeutic protein, e.g., a therapeuticpolypeptide.

In some embodiments, the vehicle formulation, e.g., the vehicle onlyformulation, substantially comprises a surfactant (e.g., poloxamer 188),a tonicity agent (e.g., sorbitol), and a buffering agent (e.g., sodiumcitrate). In embodiments, the vehicle formulation is substantially freeof protein. In embodiments, the vehicle formulation is substantiallyfree of a therapeutic protein or peptide. In embodiments, the vehicleformulation does not contain a viscosity agent.

Useful surfactants, tonicity agents, and buffering agents include thosedisclosed herein. In some embodiments, the surfactant is poloxamer 188,the tonicity agent is sorbitol, and the buffering agent is sodiumcitrate and/or sodium phosphate.

In embodiments, the buffering agent is present at a concentration of 20mM or less.

In some embodiments, the vehicle formulation comprises about 0.1% w/vpoloxamer 188, about 5% w/v sorbitol, and about 10 mM w/v sodiumcitrate. In embodiments, the components of a vehicle formulationdescribed herein are present in amounts that may vary around the valuesprovided herein by up to 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%. Inembodiments, the components of a vehicle formulation are present inamounts that vary around the values provided herein by 10%. In someembodiments, the vehicle formulation is an aqueous formulationconsisting of 10 mM sodium citrate, pH 6.0, 5% sorbitol (w/v), and 0.1%poloxamer 188.

In embodiments, the vehicle formulation comprises 9.5-10.5 mM, 9-11 mM,8.5-11.5 mM, 8-12 mM, 7.5-12.5 mM, 7-13 mM, 6-14 mM, or 5-15 mM sodiumphosphate or sodium citrate. In embodiments, the vehicle formulationcomprises 9.5-10.5 mM, 9-11 mM, 8.5-11.5 mM, 8-12 mM, 7.5-12.5 mM, 7-13mM, 6-14 mM, or 5-15 mM sodium citrate. In embodiments, the vehicleformulation comprises 4.75-5.25%, 4.5-5.5%, 4.25-5.75%, 4-6%,3.75-6.25%, 3.5-6.5%, 3-7%, or 2.5-7.5% w/v sorbitol. In embodiments,the vehicle formulation comprises 0.095-0.105%, 0.09-0.11%,0.085-0.115%, 0.08-0.12%, 0.075-0.125%, 0.07-0.13%, 0.06-0.14%, or0.05-0.15% w/v poloxamer 188.

In embodiments, the pH of the vehicle formulation is 5.5 to 7.5. Inembodiments, the pH is 5.5 to 6.5. In embodiments, the pH is 6 to 7. Inembodiments, the formulation comprises 8-12 mM sodium citrate, 4-6% w/vsorbitol, 0.08-0.12% w/v poloxamer 188, and has a pH of 5.5. to 7.5,e.g., a pH of 5.5 to 6.5.

In embodiments, the vehicle formulation comprises 9-11 mM sodiumcitrate, 4.5-5.5% w/v sorbitol, 0.09-0.11% w/v poloxamer 188, and has apH of 5.5. to 7.5, e.g., a pH of 5.5 to 6.5. In embodiments, the vehicleformulation comprises 7-13 mM sodium citrate, 3.5-5.5 w/v % sorbitol,0.07-0.13% w/v poloxamer 188, and has a pH of 5.5. to 7.5, e.g., a pH of5.5 to 6.5.

In embodiments, the vehicle formulation does not contain a viscosityagent.

In embodiments, the vehicle formulation comprises a viscosity agent,e.g., sodium carboxymethyl cellulose (CMC). In embodiments, the vehicleformulation comprises CMC, e.g., CMC at a concentration of 0.1-1% w/v,0.1-0.5% w/v, or 0.2-0.3 w/v %.

In some embodiments, the vehicle formulation comprises 0.1% w/vpoloxamer 188, 5% w/v sorbitol, 0.25% w/v sodium carboxymethyl celluloseand 10 mM sodium phosphate. In embodiments, the vehicle formulation hasa pH of about 6.5. In embodiments, the components of the formulation arepresent in amounts that may vary around the values provided by up to 5%,10%, 15%, 20%, 25%, or 30%. In some embodiments, the vehicle formulationconsists of 0.1% w/v poloxamer 188, 5% w/v sorbitol, 0.25% w/v sodiumcarboxymethyl cellulose and 10 mM sodium phosphate.

In embodiments, the formulation comprises 0.08-0.12% w/v poloxamer 188,4-6% w/v sorbitol, 0.2-0.3% w/v sodium carboxymethyl cellulose and 8-10mM sodium phosphate. In embodiments, the formulation has a pH of5.5-7.5, e.g., a pH of 5.5-6.5.

Administration

In some embodiments, a formulation featured herein, e.g., a formulationcontaining a therapeutic protein such as a chimeric IL-1 inhibitor or avehicle formulation, is administered topically to a subject, e.g., ahuman or other mammal such as a dog, cat, or horse, and, for exampleadministered to the eye. In general, a formulation described herein canbe administered to a subject, by any suitable method, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, intrasynovial, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural injection, intrasternal injection and infusion. Other suitablemodes of administration include topical (e.g., dermal or mucosal) orinhalation (e.g., intranasal or intrapulmonary) routes. For certainapplications, the route of administration is one of: intravenousinjection or infusion, subcutaneous injection, or intramuscularinjection. For administration to the eye, in some embodiments, the modeof administration for a formulation featured herein (e.g., a chimericcytokine formulation described herein) is topical administration to theeye, e.g., in the form of drops. Examples of devices that may containthe formulation and/or be used for adminstration of the formulationinclude simple eye droppers, squeeze bottles with or without meteringfunction, and blow/fill/seal (BFS) devices such as those manufactured byCatalent (Somerset, N.J.), multi-use devices using, for example tip-sealtechnology, silver/oligodynamic technology, sterile filters, collapsingprimary containers, and the like.

Another consideration for a formulation is minimizing sticking to thedelivery device or container. For example, the addition of surfactant,e.g., poloxamer 188 can minimize sticking of P05 to a container.

An additional consideration for a container is that it provide anacceptable shelf-life once it is filled, e.g., there is an acceptablylow level of evaporation and/or the formulation meets release assayspecifications, e.g., specifications as described herein. Inembodiments, the container is suitable to provide a shelf-life of atleast two years, e.g., at least 3 years, at least 4 years, or at least 5years, e.g., at 5° C. In embodiments, the container is suitable toprovide a shelf-life of at least 3 years at 5° C. In embodiments, thecontainer is suitable to provide a shelf-life of at least 2 months, 3months, 4 months, 5 months, 6 months, 8 months, 10 months, or 12 monthsat RT. In embodiments, the the container is suitable to provide ashelf-life of at least 5 months at RT. Various suitable containermaterials are known in the art, for example certain plastics, forexample, low density polyethylene (LDPE), high densidy polyethylene(HDPE), or polypropylene.

The formulation can be prepared for single use application in acontainer or can be prepared for use in a multiuse container.

A formulation featured herein can be delivered intravitreally, e.g., totreat disorders that are associated with, for example, the posteriorsegment of the eye. Methods of intravitreal administration are known inthe art and include, for example, intraocular injection, implantabledevices.

In embodiments, the formulation is administered intravitreally using animplantable device. In embodiments, the formulation comprises a thermalstabilizer, e.g., sorbitol. In embodiments, the sorbitol is present at aconcentration of ≧5% w/v.

Implantable devices can be, for example, nonbiodegradable devices suchas polyvinyl alcohol-ethylene vinyl acetate polymers and polysulfonecapillary fibers, biodegradable devices such as polylactic acid,polyglycolic acid, and polylactic-co-glycolic acid, polycaprolactones,and polyanhydrides. Devices can be delivered in forms such asnanoparticles, liposomes, or microspheres.

A formulation featured in the invention can be administered as a fixeddose, as weight determined dose (e.g., mg/kg), or as an age determineddose. The formulations, e.g., a vehicle formulation or a therapeuticformulation (a formulation that includes a therapeutic such as atherapeutic protein) can be administered, for example, four times a day;three times a day; twice a day; once every day; every other day; everythird, fourth or fifth day; every week; every two weeks; every threeweeks; every four weeks; every five weeks; monthly; every two months;every three months; every four months; every six months; or as needed(ad libitum).

In embodiments, the formulation is administered once, twice, or threetimes a day. In some such embodiments, the formulation is administeredtopically, e.g., to the surface of the eye. A pharmaceutical compositioncan include a “therapeutically effective amount” of an agent describedherein. A therapeutically effective amount of an agent can varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the compound to elicit a desiredresponse in the individual, e.g., amelioration of at least one disorderparameter (e.g., sign), or amelioration of at least one symptom of thedisorder (and optionally the effect of any additional agents beingadministered). A therapeutically effective amount is also one in whichany toxic or detrimental effects of the composition are outweighed bythe therapeutically beneficial effects. In some embodiments, a“therapeutically effective amount” is determined in a population ofindividuals and the amount is effective in ameliorating at least onesymptom or indication of a cytokine-related disorder, e.g., anIL-1-related disorder in at least 5%, 10%, 25%, 50%, 70%, 75%, 80%, 85%,90%, 95%, or 100% of an affected population. A formulation is typicallyadministered in a therapeutically effective amount. In some cases, atherapeutically effective formulation is a vehicle formulation. In somecases, a therapeutically effective formulation comprises a therapeuticprotein.

In some embodiments, the formulation is administered to a subject havingan IL-1-related disorder and the chimeric cytokine polypeptide comprisesfragments of IL-1β and Il-1Ra sequences. Such a formulation contains,for example, 5 mg/ml to 20 mg/ml, 5 mg/ml or 20 mg/ml of thepolypeptide. In embodiments, the formulation is administered topicallyto the eye once, twice, three, four, five, or six times per day.Pharmaceutical compositions can be administered using medical devices asdescribed herein and as known in the art, e.g., implants, infusionpumps, hypodermic needles, and needleless hypodermic injection devices.A device can include, e.g., one or more housings for storingpharmaceutical compositions, and can be configured to deliver unit dosesof the chimeric cytokine polypeptide, and optionally a second agent. Thedoses can be fixed doses, i.e., physically discrete units suited asunitary dosages for the subjects to be treated; each unit can contain apredetermined quantity of chimeric cytokine polypeptide calculated toproduce the desired therapeutic effect in association with apharmaceutical carrier and optionally in association with another agent,e.g., Restasis® or artificial tears such as those available as over thecounter or prescribed products.

In some embodiments, to treat a disorder described herein such as anIl-1-related disorder, the formulation is administered to a subjecthaving the disorder in an amount and for a time sufficient to induce asustained improvement in at least one sign or symptom of the disorder.An improvement is considered “sustained” if the subject exhibits theimprovement over a prolonged period, e.g., on at least two occasionsseparated by one to four weeks. The degree of improvement can bedetermined based on signs or symptoms, and can also employquestionnaires that are administered to the subject, such asquality-of-life questionnaires. In one non-limiting example, thechimeric cytokine polypeptide comprises fragments of an IL-1β and anIL-1Ra and is topically administered at least once per week, e.g., atleast once per day, at least twice per day, or at least three times perday.

Improvement can be induced by repeatedly administering a dose of theformulation until the subject manifests an improvement over baseline forselected signs and/or symptoms. In treating chronic conditions, theamount of improvement can be evaluated by repeated administration over aperiod of at least a month or more, e.g., for one, two, or three monthsor longer, or indefinitely. In treating an acute condition, the agentcan be administered for a period of one to six weeks or even as a singledose.

Although the extent of the disorder after an initial or intermittenttreatment can appear improved according to one or more signs orsymptoms, treatment can be continued indefinitely at the same level orat a reduced dose or frequency. Treatment can also be discontinued,e.g., upon improvement or disappearance of signs or symptoms. Oncetreatment has been reduced or discontinued, it may be resumed ifsymptoms should reappear.

Treatments

Some formulations featured herein comprise a therapeutic protein. Inembodiments, the formulations comprise a chimeric receptor binding agent(e.g., a chimeric cytokine) such as one that can bind to an IL-1R andthat can antagonize IL-1 signaling, and therefore can be used to treatan “IL-1 related disorder,” which includes any disease or medicalcondition that is (i) caused at least in part by IL-1 agonism, (ii) isassociated with elevated levels or activity of an IL-1 signalingcomponent (such as IL-1α, IL-1β, or IL-1RI) or elevated IL-1 signaling,and/or (iii) is ameliorated by decreasing IL-1 activity. IL-1 relateddisorders include acute and chronic disorders, including autoimmunedisorders and inflammatory disorders. IL-1 related disorders includesystemic and non-systemic disorders. It is well established that IL-1αand IL-1β are potent pro-inflammatory cytokines implicated in infectiousresponses as well as in inflammatory disease, including, e.g.,rheumatoid arthritis. Increased IL-1 production has been observed inpatients with certain autoimmune disorders, ischemia, and variouscancers, therefore implicating IL-1 in these and related diseases (forexample, see Sims and Smith, Nature Rev Immunol, 10:89-102 (2010)).

As used herein, the term “treat” refers to the administration of anagent described herein to a subject, e.g., a patient, in an amount,manner, and/or mode effective to improve a condition, symptom, orparameter associated with a disorder, e.g., a disorder described herein,or to prevent the onset or progression of a disorder, to either astatistically significant degree or to a degree detectable to oneskilled in the art. The treatment can be to cure, heal, alleviate,relieve, alter, remedy, ameliorate, palliate, improve or affect thedisorder, the symptoms of the disorder or the predisposition toward thedisorder. An effective amount, manner, or mode can vary depending on thesubject and may be tailored to the subject. Exemplary subjects includehumans, primates, and other non-human mammals. A formulation featured inthe invention can also be given prophylactically to reduce the risk ofthe occurrence of a disorder or symptom or sign thereof.

The IL-1-related disorder can be an autoimmune disorder. Examples ofIL-1-related autoimmune disorders include rheumatoid arthritis,ankylosing spondylitis, Behçet's syndrome, inflammatory bowel diseases(including Crohn's disease and ulcerative colitis), asthma, psoriasis,type I diabetes, some forms of acne, and other disorders identifiedherein. The formulations described herein can be administered to asubject having or at risk for such IL-1 mediated autoimmune disorders.The IL-1 mediated disorder can be an inflammatory disorder such asdescribed below. The formulations described herein can be administeredto a subject having or at risk for such IL-1 mediated inflammatorydisorders.

The formulations featured in the invention are particularly suited foruse in ocular disorders, e.g. ocular disorders in which it is desired toadminister the chimeric cytokine receptor directly to the eye, orlocally to the area of the eye. Exemplary IL-1-related ocular disordersinclude Sjögren's syndrome (e.g., keratoconjunctivitis sicca associatedwith Sjögren's syndrome), dry eye disorders includingkeratoconjunctivitis sicca (Sjögren's-associated ornon-Sjögren's-associated), keratitis sicca, sicca syndrome,xerophthalmia, tear film disorder, decreased tear production, aqueoustear deficiency, dry eye associated with graft-versus-host disease, andMeibomian gland dysfunction. Subjects having a dry eye disorder canexhibit inflammation of the eye, and can experience scratchy, stingy,itchy, burning or pressured sensations, irritation, pain, and redness.Dry eye disorders can be associated with excessive eye watering andinsufficient tear production. A formulation featured in the inventioncan be administered to such a subject to ameliorate or prevent the onsetor worsening of one or more such symptoms. A formulation featured in theinvention can also be used to mitigate pain, e.g., ocular pain, such aspain due to neuroinflammation, in a subject.

The embodiments described herein include methods of treating animalshaving IL-1-related disorders, for example, dry eye disorders. Dry eyecan be a serious disorder in, for example canines. Non-limiting examplesof disorders in dogs associated with dry eye include congenitaldisorders, infections (e.g., canine distemper virus), drug induction(e.g., by sulfa antibiotics), and removal of the tear gland of the thirdeyelid (“cherry eye”). Dry eye disorders are also commonly seen incertain dog breeds, for example, Cocker Spaniel, Shih Tzu, Lhasa Apso,Bulldog, Schnauzer, and West Highland White Terrier. Other non-limitingexamples of animals that can be treated include cats and horses.

The formulations featured herein can also be used to treat otherdisorders affecting the surface of the eye, such as the cornea. Suchdisorders include corneal ocular surface inflammatory conditions,corneal neovascularization, keratitis, including peripheral ulcerativekeratitis and microbial keratitis. The formulations can be used to treata subject undergoing corneal wound healing (e.g., a subject having acorneal wound). The formulation can be administered to a subject who isabout to receive, undergoing, or recovering from a procedure involvingthe eye, e.g., corneal transplantation/keratoplasty, keratoprosthesissurgery, lamellar transplantation, selective endothelialtransplantation. See, e.g., Dana (2007) Trans Am Ophthalmol Soc 105:330-43; Dekaris et al. (1999) Curr Eye Res 19(5): 456-9; and Dana et al.(1997) Transplantation 63:1501-7.

The formulation can be used to treat disorders affecting theconjunctiva, including conjunctival scarring disorders andconjunctivitis, e.g., allergic conjunctivitis, for example, severeallergic conjunctivitis. The formulation can be used to treat stillother disorders such as pemphigoid syndrome and Stevens-Johnsonsyndrome. The formulations featured in the invention can be administeredto a subject to modulate neovascularization in or around the eye. See,e.g., Dana (2007) Trans Am Ophthalmol Soc 105: 330-43.

The formulations of the present invention can be administered to asubject having an allergic reaction affecting the eye, e.g., a subjectexperiencing severe allergic (atopic) eye disease such as, e.g.,allergic conjunctivitis. For example, the formulation can beadministered topically. See also, e.g., Keane-Myers et al. (1999) InvestOphthalmol Vis Sci, 40(12): 3041-6.

The formulations featured in the invention can be administered to asubject having an autoimmune disorder affecting the eye. Exemplaryautoimmune ocular disorders include sympathetic ophthalmia,Vogt-Koyanagi Harada (VKH) syndrome, birdshot retinochoriodopathy,ocular cicatricial pemphigoid, Fuchs' heterochronic iridocyclitis, andvarious forms of uveitis. The formulations can be administered to asubject to treat any of the foregoing disorders.

The formulations featured in the invention can be administered to asubject who has or is at risk for diabetic retinopathy. See, e.g.,Demircan et al. (2006) Eye 20:1366-1369 and Doganay et al. (2006) Eye,16:163-170

Uveitis. Uveitis includes acute and chronic forms and includesinflammation of one or more of the iris, the ciliary body, and thechoroid. Chronic forms may be associated with systemic autoimmunedisease, e.g., Behçet's syndrome, ankylosing spondylitis, juvenilerheumatoid arthritis, Reiter's syndrome, and inflammatory bowel disease.In anterior uveitis, inflammation is primarily in the iris (alsoiritis). Anterior uveitis can affect subjects who have systemicautoimmune disease, but also subjects who do not have systemicautoimmune disease. Intermediate uveitis involves inflammation of theanterior vitreous, peripheral retina, and ciliary body, often withlittle anterior or chorioretinal inflammation. Pan planitis results frominflammation of the pars plana between the iris and the choroid.Posterior uveitis involves the uveal tract and primarily the choroid,and is also referred to as choroiditis. Posterior uveitis can beassociated with a systemic infection or an autoimmune disease. It canpersist for months and even years. The formulations featured in theinvention can be administered to a subject to treat any of the foregoingforms of uveitis. See also e.g., Tsai et al. (2009) Mol Vis 15:1542-1552and Trittibach et al. (2008) Gene Ther. 15(22): 1478-88.

In some embodiments, the formulations featured in the invention are usedto treat a subject having or at risk for age-related maculardegeneration (AMD). The formulations can be applied topically to theeye, injected (e.g., intravitreally) or provided systemically. See,e.g., Olson et al. (2009) Ocul Immunol Inflamm 17(3):195-200.

A formulation described herein can be administered by any mode to treatan ocular disease. The agent can be delivered by a parenteral mode.Alternatively or in addition, the formulation can be delivered directlyto the eye or in the vicinity of the eye. For example, the formulationcan be administered topically or intraocularly, e.g., as describedherein.

Formulations and Methods for Ocular Delivery

Ophthalmic formulations featured in the invention can be delivered fortopical administration, e.g., for administration as a liquid drop, anointment, or a gel, or for implantation, e.g., into an anterior chamberof the eye or the conjunctival sac. Drops, such as liquid drops, can bedelivered using an eye dropper. Gels and ointments can also beadministered using a dropper. When formulated for ocular delivery, anactive agent (e.g., the chimeric cytokine protein or receptor bindingagent) can be present at 0.0001% to 0.1%, 0.001% to 5%, e.g., 0.005% to0.5%, 0.05% to 0.5%, 0.01% to 5%, 0.1% to 2% or 1% to 5% concentration.In some embodiments, the concentration is 2%, e.g., of P05. In otherembodiments, the concentration is 0.5%, e.g., of P05.

In some embodiments, the receptor binding agent, e.g., P05 is formulatedon a mg/ml basis, e.g., as described supra. For example, the activeagent, e.g., the receptor binding agent, is an IL-1 inhibitor and ispresent at a concentration of 1-50 mg/ml, 1-25 mg/ml, 1-20 mg/ml, 1-10mg/ml, 2-8 mg/ml, 3-7 mg/ml, or 4-6 mg/ml. In embodiments, the activeagent is present at a concentration of 1 mg/ml, 2 mg/ml, 3 mg/ml, 4mg/ml, 5 mg/ml, 8 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30mg/ml, 40 mg/ml, or 50 mg/ml. In embodiments, the active agent, e.g.,the IL-1 inhibitor, is present at a concentration of up to 100 mg/ml.

Typically, the ophthalmic formulation is applied directly to the eyeincluding onto the cornea, the eyelid or instillation into the space(cul-de-sac) between the eyeball and the eyelids. The ophthalmicformulation can be designed to mix readily with the lacrimal fluids andspread over the surfaces of the cornea and conjunctiva. With the usualtechnique of administration, the major portion of the drug is typicallydeposited in the lower fornix. Capillarity, diffusional forces, and theblinking reflex drive incorporation of the drug in the precorneal filmfrom which it penetrates into and through the cornea.

Ophthalmic formulations featured in the invention can also include oneor more other agents, e.g., an anti-inflammatory steroid such asrimexolone, loteprednol, medrysone and hydrocortisone, or anon-steroidal anti-inflammatory. For example, the steroid can be presentat a concentration of 0.001% to 1%. In some embodiments, no steroid ispresent. For example, the receptor binding agent is the only activeagent in the formulation.

The formulation can also include one or more of the following componentsas described herein: surfactants, tonicity agents, buffers,preservatives, co-solvents and viscosity building agents. Tonicityagents can be used to adjust the tonicity of the composition, e.g., tothat of natural tears. Tonicity agents, particularly sugars, may alsofunction as thermal stabilizers. In embodiments, potassium chloride,sodium chloride, magnesium chloride, calcium chloride, dextrose and/ormannitol may be added to achieve an appropriate tonicity, e.g.,physiological tonicity. Tonicity agents can be added in an amountsufficient to provide an appropriate osmolality as described herein. Inembodiments, a tonicity agent is added to provide an osmolality of about150 mOsm per kg to 450 mOsm per kg or 250 mOsm per kg to 350 mOsm perkg. In embodiments, a tonicity agent is added to provide an osmolalitythat is isotonic in the eye. In embodiments, a tonicity agent, e.g.,sorbitol, is added to provide an osmolality of 270-330 mOsm per kg.

The formulation can also include buffering suitable for ophthalmicdelivery and as described herein. The buffer can include one or morebuffering components such as a citrate, phosphate, borate, boric acid,succinate, acetate or a pharmaceutically acceptable salt thereof (e.g.,sodium phosphate, sodium acetate, sodium citrate, sodium borate, sodiumsuccinate, or sodium acetate), to changes in pH. The buffering componentcan be used especially under storage conditions, e.g., when theformulation will be subjected to prolonged storage. For example, thebuffer can be selected to provide a target pH within the range of pH5.5-6.5, pH 5.5-6.0, pH 6.0 to 7.5, or pH 6.5 to 7.5. Typically, thebuffering agent is a weak buffering agent, wherein the concentration ofthe buffering components is below 20 mM. In embodiments, theconcentration of the buffering components is between about 5 to 20 mM,e.g., 5 to 15 mM, e.g., 5 to 10 mM.

The formulation comprising a therapeutic protein can include an aqueousor phospholipid carrier. Particularly for treating dry eye disorders,the formulation can include agents to provide short-term relief, e.g.,compounds that lubricate the eye and assist in tear formation. Forexample, phospholipid carriers (which include one or more phospholipids)can be used to provide short-term relief. Examples or artificial tearscompositions useful as artificial tears carriers include commercialproducts such as Tears Naturale® (Alcon Labs, Inc., TX USA). Forexample, per ml, the formulation can include: 1 mg dextran, 70 and 3 mghydroxypropyl methylcellulose, and optionally a preservative suchPOLYQUAD® (polyquaternium-1) 0.001% (m/v). Examples of phospholipidcarrier formulations include those disclosed in U.S. Pat. No. 4,804,539,U.S. Pat. No. 4,883,658, U.S. Pat. No. 5,075,104, U.S. Pat. No.5,278,151, and U.S. Pat. No. 5,578,586.

The formulation can also include other compounds that act as a lubricantor wetting agent. These include viscosity agents such as: monomericpolyols, such as, glycerol, propylene glycol, ethylene glycol; polymericpolyols, such as polyethylene glycol, various polymers of the cellulosefamily: hydroxypropylmethyl cellulose (“HPMC”), sodium carboxymethylcellulose, hydroxy propylcellulose (“HPC”), dextrans, such as dextran70; water soluble proteins, such as gelatin; and vinyl polymers, such aspolyvinyl alcohol, polyvinylpyrrolidone, povidone and carbomers, such ascarbomer 934P, carbomer 941; carbomer 940, carbomer 974P. Stilladditional examples include polysaccharides, such as hyaluronic acid andits salts and chondroitin sulfate and its salts, and acrylic acidpolymers. In certain embodiments, the formulation has a viscositybetween 1 cP to 400 cP.

The formulation, e.g., a vehicle formulation, can be packaged for singleor multi-dose use, e.g., in a bottle with an associated dropper or as aset of single-use droppers.

The formulation can include one or more preservatives, e.g., to preventmicrobial and fungal contamination during use, and/or one or moredetergents, or surfactants, e.g., to solubilize proteins. Exemplarypreservatives include: benzalkonium chloride, chlorobutanol,benzododecinium bromide, methyl paraben, propyl paraben, phenylethylalcohol, edetate disodium, sorbic acid, and polyquaternium-1, and can beincluded at a concentration of from 0.001 w/v to 1.0% w/v. Typically, aformulation containing a therapeutic protein as described herein issterile yet free of preservatives.

Exemplary detergents/surfactants include Pluronics®, such as F-68;Triton® surfactants, such as Triton X-100, polysorbates, such asTween-20 and Tween-80, Elugent™, and Cremophor® polyethoxylated castoroil, as well as tyloxapol, octoxynol 40 and polyoxyl 40 stearate.

In general, detergents and/or surfactants can be included at aconcentration of from 0.001% w/v to 1.0% w/v. In some aspects, theformulation is free of detergents.

Ophthalmic packs may be used to give prolonged contact of an ophthalmicformulation with the eye. A cotton pledget is saturated with theformulation and then inserted into the superior or inferior fornix. Theformulation may also be administered by the way of iontophoresis. Thisprocedure keeps the solution in contact with the cornea in an eyecupbearing an electrode. Diffusion of the drug is effected by difference ofelectrical potential. Iontophoretic systems which have been used includeOcuphor®1 (Iomed Inc., USA); Eyegate® II Delivery Systeml (EyeGatePharma, USA); and Visulex®1 (Aciont Inc., USA). See Amo and Urtti, DrugDiscovery Today, 13:143 (2008).

Another strategy for sustained ocular delivery is the use of gelifyingagents. These materials can be delivered in a liquid form, as an eyedrop or intraocular injection. After instillation the polymer undergoesa phase change and forms a semi-solid or solid matrix that releases thedrug over prolonged period. The phase transition can be induced bychanges in the temperature, ion concentration, or pH.

For topical ocular use, the gel forming solutions, such as Timoptic®-XE1(Merck and Co. Inc., USA), which contains Gelrite® (purified anionicheteropolysaccharide from gellan gum); Pilogel®1 (Alcon, Inc.,Switzerland) eye drops contain poly(acrylic acid); and Azasite®1 (InsiteVision, USA) have been tested clinically. These materials enhance thedrug retention relative to the conventional eye drops and lead toincreased drug absorption into the eye and reduced dosing frequency. SeeAmo and Urtti, Drug Discovery Today, 13:135-143 (2008).

A formulation featured in the invention can be delivered by injection,e.g., intravitreal, periocular, or subconjunctival injection. Theformulation can be injected underneath the conjunctiva facilitatingpassage through the sclera and into the eye by simple diffusion. Theformulation can also be injected underneath the conjunctiva and theunderlying Tenon's capsule in the more posterior portion of the eye todeliver the agent to the ciliary body, choroid, and retina. Theformulation may also be administered by retrobulbar injection.

In embodiments, a formulation provided herein is administeredintravitreally. In embodiments, the formulation does not comprise CMC.

Evaluation

With respect to dry eye and other surface disorders, subjects can beevaluated using one or more of the approaches known in the art, forexample, the Ocular Surface Disease Index (OSDI), corneal andconjunctival staining, and the Schirmer test. When the OSDI is used, anegative change from baseline indicates an improvement in vision-relatedfunction and the ocular inflammatory disorders.

For corneal fluorescein staining, saline-moistened fluorescein strips or1% sodium fluorescein solution are used to stain the tear film.Typically, the entire cornea is then examined using slit-lamp evaluationwith a yellow barrier filter (#12 Wratten) and cobalt blue illumination.Staining can be graded, e.g., according to the NEI scale, the OxfordSchema, or a modified Oxford Schema. Typically, staining is gradedaccording to the NEI scale, which is a 15 point scale where the corneais divided into 5 sections (a central circular section, and 4 quadrantssurrounding the central corneal section which are referred to asinferior, superior, nasal and temporal quadrants) each of which isscored from 0-3 for punctate staining to yield a maximum possible scoreof 15.

Conjunctival staining is likewise a measure of epithelial disease orbreak in the epithelial barrier of the ocular surface. Conjunctivalstaining is performed under the slit-lamp using lissamine green.Saline-moistened strip or 1% lissamine green solution is used to stainthe tear film, and interpalpebral conjunctival staining is evaluatedmore than 30 seconds but less than two minutes later. Using white lightof moderate intensity, only the interpalpebral region of the nasal andtemporal conjunctival staining is graded, e.g., using the Oxford Schema.

The Schirmer test is performed in the presence or in the absence ofanesthesia by placing a narrow filter-paper strip (5×3.5 mm strip ofWhatman #41 filter paper) in the inferior cul-de-sac. This test isconducted in a dimly lit room. The patient gently closes his/her eyesuntil five minutes have elapsed and the strips are removed. Because thetear front will continue advancing a few millimeters after it has beenremoved from the eyes, the tear front is marked with a ball-point pen atprecisely five minutes. Aqueous tear production is measured by thelength in millimeters that the strip wets during 5 minutes. Results of10 mm or less for the Schirmer test without anesthesia and 5 mm or lessfor the Schirmer test with anesthesia are considered abnormal. Apositive change from baseline indicates improvement of one or moresymptoms of an ocular inflammatory disorder described herein.

Dry Eye Disease Models.

Efficacy of the formulations featured in the invention can be evaluatedin a mouse model for dry eye disease. Dry eye can be induced in mice bysubcutaneous injection of scopolamine and then placement of the mice incontrolled-environment chambers. By way of a specific example, normalhealthy 6 to 10 weeks old female C57BL/6 mice can be induced to have dryeye by continuous exposure to dry environment in a controlledenvironmental chamber. The chamber has low relative humidity of lessthan 30% (generally about 19%), high airflow (15 liters/minute) andconstant temperature (about 22° C.). The mice placed in the chamber arealso treated with scopolamine to inhibit tear secretion.Sustained-release transdermal scopolamine patches can be obtained fromNovartis (Summit, N.J.). One-fourth of a patch is applied to thedepilated mid-tail of mice every 48 hours. The combination of thecontrolled environmental chamber and scopolamine produces severe dry eyein a relative short period of time (about 2-4 days). The controlledenvironmental chamber can be prepared as described in Barbino et al.(Invest Ophthal Vis Sci, 46: 2766-2711 (2005)), and enables control ofair flow, humidity, and temperature.

Mice can be monitored for signs of dry eye, e.g., by performing: a)cotton thread test to measure aqueous tear production, which isgenerally decreased in patients with dry eye; b) corneal fluoresceinstaining which is a marker of corneal surface damage; and generalophthalmic examination.

Cotton Thread Test: Tear production can be measured with cotton threadtest, impregnated with phenol red (Zone-Quick, Lacrimedics, Eastsound,Wash.). Under a magnifying fluorescent lamp, the thread is held withjeweler forceps and placed in the lateral cantus of the conjunctivalfornix of the right eye for 30 or 60 seconds. The tear distance in mm isread under a microscope using the scale of a hemacytometer.

Corneal Fluorescein Staining: Corneal fluorescein staining can beevaluated by applying 1.0 ml of 5% fluorescein by a micropipette intothe inferior conjunctival sac of the eye. The cornea is examined with aslit lamp biomicroscope using cobalt blue light 3 minutes after thefluorescein instillation. Punctuate staining is recorded in a maskedfashion using a standardized National Eye Institute (NEI) grading systemof 0-3 for each of the five areas in which the corneal surface has beendivided.

EQUIVALENTS

All technical features can be individually combined in all possiblecombinations of such features.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

The entire content of all references cited herein is hereby incorporatedin its entirety.

The following non-limiting examples further illustrate embodiments ofthe inventions described herein.

EXAMPLES Example 1 Examples of Therapeutic Proteins, e.g., ChimericProteins

Nucleic acids encoding the proteins with the amino acid sequences listedin Table 1 (below) were constructed in a pET vector containing a T7promoter and ampicillin (pET31 series) or kanamycin resistance genes(pET28 series) (EMD Chemicals, Gibbstown, N.J., USA), and expressed.Examples of coding sequences that can be used for expression areprovided in Table 2.

TABLE 1 SEQ  ID  Exemplary chimeric proteins NO: P01APVRSLAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV 1SFVQGEESNDKIPVALGIHGGKMCLSCVKSGDETRLQLEAVDPKNYPKKKMDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQ PVSLTNMPDEGVMVTKFYMQFVSS P02APVRSLAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV 2SFVQGEESNDKIPVALGIHGGKMCLSCVKSGDETRLQLEAVDPKNYPKKKMEKRFVFNKIEINNKLSFESAACPGWFLCTAMEADQ PVSLTNMPDEGVMVTKFYMQFVSS P03APVRSLAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSM 3SFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFIRSDSGPTTSFESAACPGWFLCTAMEADQ PVSLTNMPDEGVMVTKFTMQFVSS P04APVRSLAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSM 4SFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAACPGWFLCTAMEADQ PVSLTNMPDEGVMVTKFTMQFVSS P05APVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSM 5SFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCTAMEADQ PVSLTNMPDEGVMVTKFYMQFVSS P06APVRSLNCTLWDVNQKTFYLRNNQLVAGYLQGPNVEQQVVFSM 6SFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSMEADQ PVFLGGTKGGQDITDFTMQFVSS P07APVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFS 7MSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCTAME ADQPVSLTNMPDEGQDITDFTMQFVSSExemplary nucleic acid sequences encoding the above proteins are listedin Table 2. In some embodiments, the nucleic acid sequence furtherincludes an ATG prior to the first nucleotide listed below. In someembodiments, the nucleic acid sequence further includes a stop codon(such as TAA, TAG, or TGA) after the last nucleotide listed below.

TABLE 2 SEQ  Nucleic acids encoding exemplary ID chimeric proteins NO:P01 GCACCTGTACGATCACTGGCCTTCAGAATCTGGGATGTTAAC  8CAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTGATCCCAAAAATTACCCAAAGAAGAAGATGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGCGGCCCCACCACCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCC P02GCACCTGTACGATCACTGGCCTTCAGAATCTGGGATGTTAAC  9CAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTGATCCCAAAAATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCC P03GCACCTGTACGATCACTGGCCTTCAGAATCTGGGATGTTAAC 10CAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCTCCATGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCATCCGCTCAGACAGCGGCCCCACCACCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCACCATGCAATTTGTGTCTTCC P04GCACCTGTACGATCACTGGCCTTCAGAATCTGGGATGTTAAC 11CAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCTCCATGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCACCATGCAATTTGTGTCTTCC P05GCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAACC 12AGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCTCCATGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACATGCAATTTGTGTCTTCC P06GCACCTGTACGATCACTGAACTGCACGCTCTGGGATGTTAAC 13CAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCGAGCAACAAGTGGTGTTCTCCATGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCCCAGTTCCCCAACTGGTACATCAGCACCTCTATGGAAGCTGACCAGCCCGTCTTCCTGGGAGGGACCAAAGGCGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCC P07GCACCTGTACGATCACTGAACTGCAGAATCTGGGATGTTAAC 14CAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGTTCTCCATGTCCTTTGTACAAGGAGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCTGGAATTTGAGTCTGCCCAGTTCCCCAACTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCCAGGATATAACTGACTTCACCATGCAATTTGTGTCTTCCThe proteins can include a range of different residues from IL-1β andIL-1Ra as illustrated below. Among the examples P01, P02, P03, P04, andP05, the cytokine domains can have 48-70% residues from IL-1β and 55-78%residues from IL-1Ra. Because a number of amino acid residues areconserved between the two proteins, the sum of the percentage identityto IL-1β and to IL-1Ra can be greater than 100%.

TABLE 6 IL-1β Total residues IL-1RA residues residues % IL-1β % IL-1RAP06 130 62 152 85.5 40.8 P07 113 80 153 73.9 52.3 P05 108 85 153 70.655.6 P04 104 89 153 68.0 58.2 P03 94 99 153 61.4 64.7 P02 85 108 15355.6 70.6 P01 740 119 153 48.4 77.8Other examples of therapeutic proteins include IL-1Ra (e.g., anakinra),canakinumab, gevokizumab, rilanacept, or an anti-IL-1R antibody (e.g.,as produced by Amgen).

Example 2 Expression and Purification of Chimeric Proteins

Proteins that contain a hexa-histidine tag (SEQ ID NO:23) were expressedin E. coli cells BL21(DES) strain by induction with 1 mM isopropylβ-D-1-thiogalactopyranoside (IPTG) at 37° C. for 3 hours in LB brothmedia. The cells were lysed in 20-50 mM Tris, 0.5 M NaCl, 2.5 mM EDTA,0.1% Triton X-100, pH 8.0. Lysate was dialyzed against 1.25×PBScontaining 0.1% polysorbate 80, then sterile filtered through a 0.8/0.2μm filter before being subjected to immobilized ion affinitychromatography (IMAC) using a HisTrap HP® pre-packed column (GEHealthcare, Piscataway N.J., USA). The column was equilibrated in 50 mMphosphate, 500 mM NaCl, pH 7.1, loaded, and washed with same buffer. Itwas pre-eluted with 25 mM imidazole and eluted with 125 mM imidazole insame buffer. Eluted protein was dialyzed extensively against 1.25×PBS,0.1% polysorbate 80, pH 7.4.

The protein was loaded in 20 mM sodium phosphate, 0.5 M NaCl 10 mMimidazole, pH 7.4 buffer. It was eluted with 200 mM imidazole, 20 mMsodium phosphate, 0.5 M NaCl pH 7.4 buffer. Eluted protein was dialyzedextensively against PBS, 0.1% polysorbate 80, pH 7.4, concentrated usingan Amicon Ultra® (10K) filter, and stored at 4° or −80° C.

Proteins lacking a hexa-histidine tag (SEQ ID NO:23) were purified byion exchange chromatography. P05 protein was purified by ion exchangechromatography. Lysate from expressing cells was applied to a GigaCapS™column (Tosoh Bioscience LLC, King of Prussia, Pa., USA) at low pH(approximately pH 5.5) in the absence of salt (conductivityapproximately 1 mS/cm). The column was then eluted by a pH gradient(Buffer A=10 mM acetic acid, pH 5.5; Buffer B=20 mM Tris pH 8). A 5 mlfraction containing the eluted protein was then diluted with 5 ml of H₂Oand 5 ml of 20 mM Tris pH 8) and then applied to Capto™ Q resin (GEHealthcare, Piscataway N.J., USA) and eluted with a 0 mM to 250 mM NaClgradient in 20 mM Tris pH 8.0. The eluted protein was dialyzedextensively against 1.25×PBS 0.1% TWEEN® 80 or 1.25×PBS lacking TWEEN®and stored. See FIG. 1. P03 and P04 proteins were purified using similarmethods.

Cells expressing P05 were also grown in TEKNOVA™ Terrific Broth withanimal free soytone (# T7660) supplemented with 10 g/L glucose, 10 mMMgSO₄, trace elements (1 mg/ml TEKNOVA™ 1000× Trace Elements, #T1001),and antibiotic in a Sartorius 2L BIOSTAT™ A+ and were induced at OD35-40 with 1 mM IPTG for about 6 hours. Cells were grown at 37° C. with30% dissolved oxygen at pH 7.0, and agitation at 200-800 rpm with oxygensparge at 2 L/min Cells were fed 9 g glucose/L/hr when glucose wasdepleted as detected by a pH increase. Feed was reduced to 6 gglucose/L/hr when the pH decreased (about 2.5 hrs after induction).

Cells were collected and lysed in lysis buffer (20 mM Tris, 10 mM EDTA,0.1% Triton, pH 8.0; 20 mM Tris, 10 mM EDTA, 0.1% Triton, pH 7.0; 50 mMMOPS, 10 mM EDTA, 0.1% Triton, pH 6.5; or 50 mM MOPS, 10 mM EDTA, 0.1%Triton, pH 6.0). Lysate is loaded onto Poros® XS cation ion exchangemedia (Life Technologies Corp., Carlsbad Calif. USA) at pH 5.3 and 3mS/cm (35 mg product per ml column resin).

In an exemplary procedure, P05 protein is eluted by a step to pH 7.0using buffer containing 100 mM MOPS 25 mM NaCl pH 7.0. The first elutingpeak was discarded, and the second eluting peak was collected in poolsand contained P05 protein. Early pools are enriched for intact P05protein relative to a des-Ala species. This eluted material is thenflowed over Capto®Q anion exchange resin. The flow through, whichcontains intact P05 protein, is collected.

In another exemplary procedure, the media is washed with 100 mM MOPS 20mM NaCl pH 6.0. P05 protein is eluted by a step to pH 6.0 using buffercontaining 100 mM MOPS 50-58 mM NaCl pH 6.0. The first eluting peak wasseparated from subsequent peaks and contained intact P05 protein. Thiseluted material is then flowed over Capto®Q anion exchange resin. Theflow through, which contains intact P05 protein, is collected.

Example 3 Cell-Based Assays

The proteins or supernatants containing the proteins were evaluated in acell-based assay for IL-1 activity. HEK-Blue™ IL-1β responsive cellswere used to monitor IL-1β activity (available from InvivoGen Inc., SanDiego Calif., USA). These cells include a secreted embryonic alkalinephosphatase (SEAP) reporter gene under the control of the IFN-β minimalpromoter fused to five NF-kB and five AP-1 binding sites. IL-1βengagement of IL-1 receptors on the cell surface led to NF-kB activationand SEAP production. The SEAP report can be detected, e.g., usingQUANTI-Blue™ (InvivoGen Inc., San Diego Calif., USA) andspectrophotometric analysis. A HEK-Blue IL-1β cell suspension wasprepared from cells cultured to 70-80% confluence. The resuspended cellswere adjusted to −330,000 cells/ml in fresh growth medium (DMEM, 4.5 g/lglucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum(30 minutes at 56° C.), 50 U/mL penicillin, 50 mg/ml streptomycin, 100mg/ml Normocin®T).

Reagents were added to wells of a flat-bottom 96-well cell cultureplate: 10 μl of IL-1β at 20 ng/ml, 10 μl of the agent of interest, and30 μl of cell culture medium to a final volume of 50 μl. Positive andnegative control samples were prepared in parallel. Then 150 μl ofHEK-Blue IL-1β cell suspension (50,000 cells) was added to each well andthe plate was cultured overnight at 37° C. in 5% CO₂ tissue cultureincubator. Generally, the final IL-1β concentration (in the 200 μl finalvolume) was 0.1 ng/ml. IL-1β activity was evaluated the next day (12-15hours later). Prior to quantitation, the QUANTI-Blue™ reagent wasprepared according to the manufacturer's instructions. A flat bottomed96-well assay plate was prepared in which 150 μl of QUANTI-Blue™solution was added to each well. 50 μl of conditioned media from thewells of the 96 well tissue culture plate was added to each well of theassay plate. The plate was incubated at 37° C. for approximately 15-20minutes. SEAP levels were then measured using a spectrophotometer at620-655 nm.

Results.

As shown in FIG. 2A, in this assay, the P06 protein behaved as an IL-1RIagonist, the P07 protein behaved as a partial agonist, and the P01protein failed to agonize. In fact, the P01 protein behaved as anantagonist when assayed in the presence of IL-1β. FIG. 2B showsantagonism of IL-1β activity by P01 at a range of IL-1β proteinconcentrations using the HEKBlue™ cell assay described herein.Antagonism increased with increasing amounts of P01 (x-axis reflectsmicroliters of supernatant containing P01).

The proteins P01, P02, P03, P04, and P05 each antagonized IL-1βactivity. See FIG. 3A and FIG. 3B, for example. The IC50 of P05 was lessthan about 5 ng/ml. P05 was test for ability to agonize IL-1RI in thisassay and was not observed to have any detectable agonistic activityeven at the highest concentrations tested, 1 mg/ml. P01, P02, P03, P04,and P05 also inhibited IL-1β induced IL-6 expression in MG-63 cells, ahuman osteosarcoma cell line that is responsive to IL-1β. In a murinemodel of dry eye disease, hexa-histidine tagged (SEQ ID NO:23) P05 wasobserved to have biological activity. See also Example 9 below regardinguntagged P05.

Example 4 Binding Properties of Chimeric Proteins

The binding properties of proteins for soluble recombinant human IL-1RI(corresponding to the extracellular domain of IL-1RI) were evaluatedusing surface plasmon resonance with a Reichert SR7000DC Dual ChannelSPR system. Binding was evaluated in phosphate buffered saline with0.005% Tween 20. IL-1β was observed to have a K_(D) of between 8-9 nMand a dissociation constant (K_(d)) of between 2-3×10⁻³ s⁻¹, and inanother experiment a K_(D) of about 2 nM, an association constant of1.3-1.5×10⁶ M⁻¹s⁻¹, and a dissociation constant (K_(d)) of about2.9-3.0×10⁻³ s⁻¹. The P01 protein bound with similar associationkinetics as IL-1β, but did not dissociate during of the dissociationphase of the binding experiment (about 180 seconds). Thus, the P01protein bound to IL-1RI with a greater affinity than did IL-1β undersimilar conditions.

Binding of IL-1Ra was observed to have a K_(D) of about 0.33 nM, anassociation constant (K_(a)) of about 2×10⁵ M⁻¹s⁻¹, and a dissociationconstant (K_(d)) of about 6.6×10⁻⁵ s⁻¹. Chimeric cytokine domains P01,P02, P03, P04, and P05 were observed to have K_(D) ranging from about12-1700 pM, an association constant (K_(a)) ranging from about 3×10⁴M⁻¹s⁻¹ to 3×10⁶ M⁻¹s⁻¹, and a dissociation constant (K_(d)) ranging fromabout 2×10⁻⁵ to 1×10⁻³ s⁻¹. See Table 3 below.

TABLE 3 Protein k_(a) (M⁻¹s⁻¹) K_(d) (s⁻¹) K_(D) (pM) IL-1β 1.47 × 10⁶M⁻¹s⁻¹ 2.95 × 10⁻³ s⁻¹ 2010 IL-1Ra 2.01 × 10⁵ M⁻¹s⁻¹ 6.58 × 10⁻⁵ s⁻¹ 326P01 4.93 × 10⁴ M⁻¹s⁻¹ 2.32 × 10⁻⁵ s⁻¹ 470 P02 3.39 × 10⁴ M⁻¹s⁻¹ 2.16 ×10⁻⁵ s⁻¹ 636 P03  4.1 × 10⁶ M⁻¹s⁻¹  1.2 × 10⁻³ s⁻¹ 290 P04 3.00 × 10⁴M⁻¹s⁻¹ 5.14 × 10⁻⁴ s⁻¹ 1714 P05 3.47 × 10⁶ M⁻¹s⁻¹ 4.15 × 10⁻⁵ s⁻¹ 12 P06 4.8 × 10⁶ M⁻¹s⁻¹  1.7 × 10⁻³ s⁻¹ 410 P07 1.58 × 10⁴ M⁻¹s⁻¹ 1.46 × 10⁻³s⁻¹ 92553

Example 5 Additional Examples of Chimeric Proteins

Additional exemplary chimeric IL-1 family proteins also include thefollowing:

P08 SEQ ID NO: 15 APVRSLAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCT AMEADQPVSLTNMPDEGVMVTKFYMQFVSSP09 SEQ ID NO:16 APVRSQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCT AMEADQPVSLTNMPDEGVMVTKFYMQFVSSP10 SEQ ID NO: 17 APVRSLAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCT AMEADQPVSLTNMPDEGVMVTKFYMQFVSSP11 SEQ ID NO: 18 APVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCT AMEADQPVSLTNMPDEGVMVTKFYMQFVSSP12 SEQ ID NO: 19 APVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCT AMEADQPVSLTNMPDEGVMVTKFTMQFVSSP13 SEQ ID NO: 20 APVRSLAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCT AMEADQPVSLTNMPDEGVMVTKFYFQEDP14 SEQ ID NO: 21 APVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWFLCT AMEADQPVSLTNMPDEGVMVTKFYFQED

The polypeptide below is a chimeric domain that includes at least twosegments from IL-1α and at least two segments from IL-1Ra.

SEQ ID NO: 22 SAPFSFLSNVKYNFMRIIKYEFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFDMGAYKSSKDDAKITVILRISKTQLYVTAQDEDQPVLLKEMPEIPKTITGSETNLLFFWETHGTKNYFTSVAHPNLFLCTAMEADQPVSLTNMPDEGVMVTKFYILENQA

Example 6 Formulation Example

An exemplary formulation according to the present invention is describedas follows:

A formulation having P05 protein present in a concentration of 25 g/l;carboxymethylcellulose is present in a concentration of 0.25% w/v;poloxamer 188 is present in a concentration of 0.1% w/v; sorbitol ispresent in a concentration of 5% w/v; sodium phosphate is present in aconcentration of 10 mM; arginine and/or glutamic acid are present in aconcentration of 100 mM. The formulation has a pH of 6.5. Theformulation is tested for stability at two weeks at room temperature andup to at least twelve months storage stability at 2-8° C. as measuredusing one or more of reverse phase HPLC (RP-HPLC); weak cation exchangeHPLC (WCEX-HPLC); spectrophotometry (A280); and visual assays.

Example 7 Formulation Example and Stability Studies

Formulations of P05 (also known as EBI-005) utilized in Phase 1 clinicalstudies were aqueous formulations that contained sodiumcarboxymethylcellulose in a concentration of 0.25% w/v; poloxamer 188 ina concentration of 0.1% w/v; sorbitol in a concentration of 5% w/v;sodium phosphate in a concentration of 10 mM, and P05 in a concentrationof either 5 or 20 mg/mL. The formulation has a pH of about 6.5. Theseformulations were tested for stability with the following measurements:appearance, pH, osmolality, content by spectrophotometry (A280),SDS-PAGE non-reduced, SDS-PAGE reduced; size exclusion HPLC (SE HPLC);reverse phase HPLC (RP-HPLC); WCEX-HPLC; potency; and containerintegrity (CIT). These tests were carried out (1) at release (0 months);(2) after storage at 5±3° C. for 1 month, 2 months, 3 months, 4 months,5 months, and 6 months; (3) after storage at 25° C. and 60% relativehumidity (a room temperature experiment) for 1 month or 3 months.Specifications for these measures and results from representativebatches of the 5 mg/ml and 20 mg/ml formulations are shown in Table14A-E below. These results demonstrate that the formulations hadexcellent stability; the formulations continued to satisfy thespecifications even after storage for 6 months at 2-8° C. and afterstorage at room temperature for 3 months.

TABLE 14A EBI-005 Phase 1 GMP Drug Product (20 mg/mL, Batch X1) at 5 ±3° C. Release Data Stability Time Point Results Analysis Specification 0Months 1 Month 2 Months* 3 Months 4 Months 5 Months 6 MonthsPhysio-Chemical Tests Appearance Clear to Clear to Clear to Clear toClear to Clear to Clear to Clear to slightly slightly slightly slightlyslightly slightly slightly slightly opalescent opalescent opalescentopalescent opalescent opalescent opalescent opalescent colorlesscolorless colorless colorless colorless colorless colorless colorlesssolution solution solution solution solution solution solution solutionessentially essentially essentially essentially essentially essentiallyessentially essentially free from free from free from free from freefrom free from free from free from visible visible visible visiblevisible visible visible visible particulates particulates particulatesparticulates particulates particulates particulates particulates pH 6.2to 6.8 6.5 6.6 6.6 6.6 6.6 6.5 6.3 Osmolality 270 to 370 328 NS NS NS NSNS NS mOsm/kg mOsm/kg Content Content 20 ± 2.0 18.3 18.4 18.2 18.4 18.418.7 18.5 by A280 mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mLSuperseded by 4 month Identity SDS- Main band Main band Main band Mainband Main band Main band Main band Main band PAGE conforms to conformsto conforms to conforms to conforms to conforms to conforms to conformsto Non- reference reference reference reference reference referencereference reference Reduced standard standard standard standard standardstandard standard standard SDS- Main band Main band Main band Main bandMain band Main band Main band Main band PAGE conforms to conforms toconforms to conforms to conforms to conforms to conforms to conforms toReduced reference reference reference reference reference referencereference reference standard standard standard standard standardstandard standard standard Purity SE-HPLC >90% (a/a) 99% (a/a) 99% (a/a)100% (a/a) 100% (a/a) 100% (a/a) 100% (a/a) 100% (a/a) Monomer MonomerMonomer Monomer Monomer Monomer Monomer Monomer RP-HPLC ≧75% (a/a) 93%(a/a) 95% (a/a) 93% (a/a) 93% (a/a) 93% (a/a) 93% (a/a) 93% (a/a) MainPeak Main Peak Main Peak Main Peak Main Peak Main Peak Main Peak MainPeak WCEX- ≧85% (a/a) 96% (a/a) 95% (a/a) 94% (a/a) 94% (a/a) 95% (a/a)95% (a/a) 94% (a/a) HPLC Main Peak Main Peak Main Peak Main Peak MainPeak Main Peak Main Peak Main Peak <10% (a/a) 4% (a/a) 4% (a/a) 5% (a/a)5% (a/a) 4% (a/a) 4% (a/a) 4% (a/a) des-Ala- des-Ala- des-Ala- des-Ala-des-Ala- des-Ala- des-Ala- des-Ala- EBI-005 EBI-005 EBI-005 EBI-005EBI-005 EBI-005 EBI-005 EBI-005 Meth Report 0.1% (a/a) 0.1% (a/a) 0.2%(a/a) 0.2% (a/a) 0.2% (a/a) 0.2% (a/a) 0.3% (a/a) Result MethionatedMethionated Methionated Methionated Methionated Methionated MethionatedAcet Report 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Result AcetylatedAcetylated Acetylated Acetylated Acetylated Acetylated AcetylatedActivity Potency IC₅₀ 50- 82% 93% 107% 94% 92% 173% 173% Eleven 200% of(used 6 QC-007 reference month data) standard IC₅₀ Sterility Endotoxin≦5.6 <1 NS NS NS NS NS NS EU/mL EU/mL Integrity Testing CIT No Pass NSNS NS NS NS NS Ingress of Dye NS = Not Sampled *Optional time point at12 months changed to a 2 month time point

TABLE 14B EBI-005 Phase 1 GMP Drug Product (20 mg/mL, Batch X1) at 25°C./60% Relative Humidity Stability Time Point Results AnalysisSpecification 1 Month 3 Months Physio-Chemical Tests Appearance Clear toslightly Clear to slightly Clear to slightly opalescent opalescentcolorless opalescent colorless colorless solution solution essentiallyfree solution essentially essentially free from visible from visibleparticulates free from visible particulates particulates pH 6.2 to 6.86.6 6.6 Osmolality 270 to 370 mOsm/kg NS NS Content Content by A280 20 ±2.0 mg/mL 18.4 mg/mL 19.5 mg/mL Identity SDS-PAGE Non- Main bandconforms to Main band conforms Main band conforms to Reduced referencestandard to reference standard reference standard SDS-PAGE Reduced Mainband conforms to Main band conforms Main band conforms to referencestandard to reference standard reference standard Purity SE-HPLC >90%(a/a) Monomer 99% (a/a) Monomer 99% (a/a) Monomer RP-HPLC ≧75% (a/a)Main Peak 94% (a/a) Main Peak 90% (a/a) Main Peak WCEX-HPLC ≧85% (a/a)Main Peak 92% (a/a) Main Peak 86% (a/a) Main Peak <10% (a/a) des-Ala- 4%(a/a) des-Ala-EBI- 4% (a/a) des-Ala-EBI-005 EBI-005 005 0.1% (a/a)Methionated Meth Report Result 0.1% (a/a) 0.0% Acetylated Acet ReportResult Methionated 0.0% Acetylated Activity Potency IC₅₀ 50-200% of 100%94% Eleven QC-007 reference standard IC₅₀ Sterility Endotoxin ≦5.6 EU/mLNS NS Integrity Testing CIT No Ingress of Dye NS NS NS = Not Sampled

TABLE 14C EBI-005 Phase 1 GMP Drug Product (20 mg/mL, Batch X2) at 5 ±3° C. Release Data Stability Time Point Results Analysis Specification 0Months 1 Month 2 Months* 3 Months 4 Months Appearance Clear to Clear toNS Clear to NS Clear to slightly slightly slightly slightly opalescentopalescent opalescent opalescent colorless colorless colorless colorlesssolution solution solution solution essentially essentially essentiallyessentially free from free from free from free from visible visiblevisible visible particulates particulates particulates particulates pH6.2 to 6.8 6.6 NS 6.6 NS 6.4 Osmolality 270 to 370 326 NS NS NS NSmOsm/kg mOsm/kg Content by 20 ± 2.0 18.5 mg/mL NS 18.9 mg/mL NS 18.7mg/mL A280 mg/mL SDS-PAGE Main band Main band NS Main band NS Main bandNon-Reduced conforms to conforms to conforms to conforms to referencereference reference reference standard standard standard standardSDS-PAGE Main band Main band NS Main band NS Main band Reduced conformsto conforms to conforms to conforms to reference reference referencereference standard standard standard standard SE-HPLC >90% (a/a) 100%(a/a) NS 100% (a/a) NS 100% (a/a) Monomer Monomer Monomer MonomerRP-HPLC ≧75% (a/a) 92% (a/a) 93% (a/a) 93% (a/a) 91% (a/a) 92% (a/a)Main Peak Main Peak Main Peak Main Peak Main Peak Main Peak WCEX-HPLC≧85% (a/a) 94% (a/a) NS 94% (a/a) NS 94% (a/a) Main Peak Main Peak MainPeak Main Peak <10% (a/a) 4% (a/a) 4% (a/a) 4% (a/a) des-Ala- des-Ala-des-Ala- des-Ala- EBI-005 EBI-005 EBI-005 EBI-005 Meth Report 0.2% (a/a)0.4% (a/a) 0.2% (a/a) Result Methionated Methionated Methionated AcetReport 0.0% 0.0% 0.0% Result Acetylated Acetylated Acetylated PotencyIC₅₀ 50- 83.0% 100.0% 100.0% 100.0% 100.0% Eleven 200% of (used 4 (used4 (used 4 QC-007 reference month data) month data) month data) standardIC₅₀ Endotoxin ≦5.6 0.3 NS NS NS NS EU/mL EU/mL CIT No Ingress of PassNS NS NS NS Dye NS = Not Sampled *Optional time point at 12 monthschanged to a 2 month time point

TABLE 14D EBI-005 Phase 1 GMP Drug Product (5 mg/mL, Batch X3) at 5 ± 3°C. Release Data Stability Time Point Results Analysis Specification 0Months 1 Month 2 Months* 3 Months 4 Months 5 Months 6 MonthsPhysio-Chemical Tests Appearance Clear to Clear to Clear to Clear toClear to Clear to Clear to Clear to slightly slightly slightly slightlyslightly slightly slightly slightly opalescent opalescent opalescentopalescent opalescent opalescent opalescent opalescent colorlesscolorless colorless colorless colorless colorless colorless colorlesssolution solution solution solution solution solution solution solutionessentially essentially essentially essentially essentially essentiallyessentially essentially free from free from free from free from freefrom free from free from free from visible visible visible visiblevisible visible visible visible particulates particulates particulatesparticulates particulates particulates particulates particulates pH 6.2to 6.8 6.5 6.6 6.5 6.5 6.6 6.4 6.3 Osmolality 270 to 370 327 NS NS NS NSNS NS mOsm/kg mOsm/kg Content Content by 5 ± 0.5 4.7 4.6 4.6 4.6 4.6 4.74.6 A280 mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL Superseded by 4month Identity SDS- Main band Main band Main band Main band Main bandMain band Main band Main band PAGE conforms to conforms to conforms toconforms to conforms to conforms to conforms to conforms to Non-reference reference reference reference reference reference referencereference Reduced standard standard standard standard standard standardstandard standard SDS- Main band Main band Main band Main band Main bandMain band Main band Main band PAGE conforms to conforms to conforms toconforms to conforms to conforms to conforms to conforms to Reducedreference reference reference reference reference reference referencereference standard standard standard standard standard standard standardstandard Purity SE-HPLC >90% (a/a) 99% (a/a) 99% (a/a) 100% (a/a) 100%(a/a) 100% (a/a) 100% (a/a) 100% (a/a) Monomer Monomer Monomer MonomerMonomer Monomer Monomer Monomer RP-HPLC ≧75% (a/a) 89.2% (a/a) 92% (a/a)82% (a/a) 82% (a/a) 84% (a/a) 76% (a/a) 91% (a/a) Main Peak Main PeakMain Peak Main Peak Main Peak Main Peak Main Peak Main Peak WCEX- ≧85%(a/a) 96% (a/a) 94% (a/a) 94% (a/a) 94% (a/a) 95% (a/a) 95% (a/a) 94%(a/a) HPLC Main Peak Main Peak Main Peak Main Peak Main Peak Main PeakMain Peak Main Peak <10% (a/a) 4% (a/a) 5% (a/a) 5% (a/a) 5% (a/a) 4%(a/a) 4% (a/a) 4% (a/a) des-Ala- des-Ala- des-Ala- des-Ala- des-Ala-des-Ala- des-Ala- des-Ala- EBI-005 EBI-005 EBI-005 EBI-005 EBI-005EBI-005 EBI-005 EBI-005 Meth Report 0.1% (a/a) 0.2% (a/a) 0.2% (a/a)0.2% (a/a) 0.2% (a/a) 0.3% (a/a) 0.3% (a/a) Result MethionatedMethionated Methionated Methionated Methionated Methionated MethionatedAcet Report 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% Result AcetylatedAcetylated Acetylated Acetylated Acetylated Acetylated AcetylatedActivity Potency IC₅₀ 50- 100% 191% 107% 115% 146% 146% 146% 200% of(used 6 (used 6 reference month data) month data) standard IC₅₀Sterility Endotoxin ≦5.6 <1 NS NS NS NS NS NS EU/mL EU/mL IntegrityTesting CIT No Pass NS NS NS NS NS NS Ingress of Dye NS = Not Sampled*Optional time point at 12 months changed to a 2 month time point

TABLE 14E EBI-005 Phase 1 GMP Drug Product (5 mg/mL, X3) at 25° C./60%Relative Humidity Stability Time Point Results Analysis Specification 1Month 3 Months Physio-Chemical Tests Appearance Clear to slightly Clearto slightly Clear to slightly opalescent opalescent colorless opalescentcolorless colorless solution solution essentially free solutionessentially essentially free from visible from visible particulates freefrom visible particulates particulates pH 6.2 to 6.8 6.6 6.5 FujifilmAM0001 Osmolality 270 to 370 mOsm/kg NS NS Content Content by A280 5 ±0.5 mg/mL 4.6 mg/mL 4.8 mg/mL Identity SDS-PAGE Non- Main band conformsto Main band conforms Main band conforms to Reduced reference standardto reference standard reference standard SDS-PAGE Reduced Main bandconforms to Main band conforms Main band conforms to reference standardto reference standard reference standard Purity SE-HPLC >90% (a/a)Monomer 99% (a/a) Monomer 100% (a/a) Monomer RP-HPLC ≧75% (a/a) MainPeak 81% (a/a) Main Peak 81% (a/a) Main Peak WCEX-HPLC ≧85% (a/a) MainPeak 93% (a/a) Main Peak 88% (a/a) Main Peak <10% (a/a) des-Ala- 4%(a/a) des-Ala-EBI- 4% (a/a) des-Ala-EBI-005 EBI-005 005 0.2% (a/a)Methionated Meth Report Result 0.2% (a/a) 0.0% Acetylated Acet ReportResult Methionated 0.0% Acetylated Activity Potency IC₅₀ 50-200% of 182%90% reference standard IC₅₀ Sterility Endotoxin ≦5.6 EU/mL NS NSIntegrity Testing CIT No Ingress of Dye NS NS NS = Not Sampled

Example 8 Melting Profiles

Proteins P03, P04, P05, mIL-1Ra (methionyl IL-1Ra), and IL-1β wereprepared in phosphate-buffered saline (PBS), pH 7.4, at 0.5 mg/ml. Theproteins were combined with SYPRO® orange dye (Invitrogen, Calif.) at a1:500 dilution of the stock concentration and subject to differentialscanning fluorimetry. See, e.g., He et al. (2010) J Pharm Sciences, 991707-1720. Fluorescence measurements were monitored using an AgilentMx3005 QPCR machine as the temperature was increased from 25° C. to 95°C. at a rate of 1° C. per minute. Melting temperature (T_(m)) valueswere derived from the maxima value of the first derivative of thefluorescence transition. The proteins P03, P04, and P05 were observed tohave an onset of unfolding of greater than 50° C. and as high as 59° C.,and T_(m) of greater than 59, 60, 62, and 64° C. Results are shown inTable 4 below and FIG. 5A and FIG. 5B:

TABLE 4 Protein T_(m) (° C.) Onset of unfolding (° C.) mIL-1Ra 56 48IL-1β 56 41 P03 65 59 P04 60 51 P05 65 59

P04 has a T_(m) that is about four degrees higher than mIL-1Ra and IL-1βand exhibits an onset of unfolding about three degrees higher thanmIL-1Ra and about ten degrees higher than IL-1β. P03 and P05 have aT_(m) that is about nine degrees higher than mIL-1Ra and IL-1β andexhibit an onset of unfolding about 11 degrees higher than mIL-1Ra andabout 18 degrees higher than IL-1β. These data demonstrate methods ofdetermining melting temperature an IL-1 inhibitor, e.g., in aformulation described herein.

Example 9 Treatment of Dry Eye in an Animal Model

Purified P05 (lacking a hexa-histidine tag (SEQ ID NO:23)) was preparedin 1.25×PBS and tested in a murine model of dry eye disease. In thismodel, female C57BL/6 mice 6 to 10 weeks of age from JacksonLaboratories (acclimated for 1 to 2 weeks in an animal holding room with≧30% relative humidity, hydrogel food supplement, and Enviro-Dri™environment enrichment) were pre-screened for fluorescein staining onDay 0. For fluorescein staining, freshly made fluorescein diluted in WFIH₂O at 10 mg/mL was administered at 0.4 μL to each eye. Approximately8-13 minutes after administration, eyes were scored using an Olympusfluorescent dissecting microscope. Punctuate staining was recorded usingthe standardized National Eye Institute (NEI) grading system of 0-3 foreach of the five areas into which the corneal surface has been divided(score range 0-15/eye). Using a teaching bridge, two masked scorersevaluated mice at the same time to give a single collective score foreach eye.

Mice with scores ≦7 for each eye (out of a maximal score of 15) wereplaced in a dry eye chamber (20%±2% humidity and constant air flow ˜21L/min/cage) on day 1 and were maintained in this chamber during thecourse of the experiment (except for examination). On day 3, mice werescored again and randomized into treatment groups with 8 to 10mice/group. Mice were randomized such that each cage of 4 to 5 mice hadapproximately the same mean disease score. Beginning on day 3 and afterrandomization, mice were topically administered P05 or vehicle(1.25×PBS) in an eye drop at 3 μL/eye BID. Mice were examined and scoredon days 7, 9, and 11 for corneal fluorescein staining as describedabove. Scorers were blinded as to the treatment groups during the courseof the experiment.

FIG. 6A is a bar graph of mean corneal staining score±SEM at day 0, 3,7, 9, and 11 for mice from two identical experiments under the followingbid treatments: no treatment, vehicle (1.25×PBS), and 10 mg/ml (1%) P05.10 mg/ml P05 significantly reduced corneal staining on days 7, 9, and 11of the experiment. Efficacy as evaluated by a reduction in cornealstaining was also observed with doses as low as 0.1 mg/ml P05.Recombinant IL-1Ra produced in E. coli also moderately reduced cornealstaining in the animal model.

As shown in FIG. 6B, the effect of 10 mg/ml P05 was specific based on acomparison to 10 mg/ml murine serum albumin in the same vehicle. Noeffect was seen with 10 mg/ml murine serum albumin (MSA) relative tovehicle, and the effect of 10 mg/ml P05 was statistically significantrelative to 10 mg/ml murine serum albumin. As shown in FIG. 6C, 10 mg/mlP05 was also compared to 0.05% cyclosporine in an ophthalmic emulsion(Restasis®). Whereas P05 reduced corneal staining, no effect wasobserved for the 0.05% cyclosporine ophthalmic emulsion after about 1week of b.i.d. dosing. These experiments demonstrate methods of testingefficacy of an IL-1 inhibitor in a formulation described herein.

Example 10 Agitation Studies

To identify a surfactant suitable for use, P05 at 1 mg/ml and at 50mg/ml was prepared in solutions of either (i) PBS, 0.5% w/v CMC, pH 7.4or (ii) 10 mM sodium citrate, pH 6.0 containing various surfactants.Agitation was performed by vortexing the protein at room temperature forfour hours. The samples were analyzed by micro-fluid imaging (MGI), SEC,A₂₈₀, and visual inspection. It was found that the use of 0.1% w/vpoloxamer 188, compared with other surfactants (including, e.g.,polysorbate 20, polysorbate 80, or no surfactant) protected the proteinfrom precipitation (assessed using visual inspection) and significantsubvisible particle accumulation during agitation. For example, the 0.1%w/v poloxamer reduced particle counts for particles ≧10 microns and forparticles ≧25 microns.

This result demonstrates that poloxamer 188 is a suitable surfactant forformulating a polypeptide such as P05. Furthermore, it demonstrates thatas little as 0.1% w/v surfactant can be effective for limiting and evendecreasing the amount of precipitation. These experiments alsodemonstrate a method of determining the suitability of a formulationdescribed herein.

Example 11 Preparation of a Formulation

Formulations of P05 were prepared. In brief, P05 was provided as afrozen liquid containing 52.8 mg/mL P05 in 1×PBS, pH 6.5. Thepolypeptide was dialyzed against a buffer containing 10 mM sodiumcitrate and 5% w/v sorbitol at pH 6.0 using 3500 molecular weight cutoffdialysis cassettes in an approximately 10,000-fold exchange over about24 hours at 2-8° C. Following dialysis, the concentration was determinedby measuring A280/A320.

After dialysis, formulations were prepared with various concentrationsof P05 as follows, 100× poloxamer 188 surfactant was added to a 1×concentration to a stock solution of dialyzed P05. The proteinconcentrations were adjusted to approximately 1 mg/mL, 5 mg/mL and 20mg/mL by adding formulation buffer (10 mM Na citrate, 5% w/v sorbitol,pH 6.0). The final concentration of the formulation components was about10 mM sodium citrate, 5% w/v sorbitol, and 0.1% w/v poloxamer 188.Samples were then mixed and sterile filtered under aseptic conditionsthen were filled (at 250 μL) into 2 cc glass vials under asepticconditions. After preparation, samples were placed in stability studiesto confirm stability of the formulation prepared under the foregoingconditions.

This demonstrates a method of preparing an IL-1 inhibitor, e.g., anIL-1beta/IL-1Ra chimeric protein formulation.

Example 12 Stability of P05 in Phosphate Versus Citrate Buffer

Dynamic light scattering or DLS (also known as quasi elastic lightscattering or QELS) measures the diffusion of an analyte (e.g., P05) ina well plate by focusing laser light on the sample, and monitoring therate of fluctuation of the scattered light as measured by a fast photoncounter. A mathematical technique, known as a correlation function, isused to quantify the rate of fluctuation to determine the diffusioncoefficient. The diffusion coefficient is used to obtain radius ofhydration (Rh) by the Stokes-Einstein equation.

The radius of P05 was measured as a function of increasing temperaturein a DLS plate reader (Wyatt DynaPro™, Wyatt Technologies, SantaBarbara, Calif.). The acquisition time was 5 seconds and 5 scans wereperformed for each measurement. The ramp rate was 0.17° C./min. As theprotein unfolded, the radius increased. The temperature at which theradius increased is referred as T_(on) (temperature of onset forunfolding).

This experiment was performed for P05 at 20 mg/mL in two formulations:(i) 10 mM phosphate, 5% w/v sorbitol, 0.1% w/v poloxamer 188, pH 6.5 and(ii) 10 mM sodium citrate, 5% w/v sorbitol, 0.1% w/v poloxamer 188, pH6.0. The results, depicted in FIG. 4A and FIG. 4B, showed that theT_(on) occurred at a much higher temperature in the citrate bufferrelative to phosphate buffer. The T_(on) was 48.2° C. for P05 in citratebuffer, and 35.2° C. for P05 in phosphate buffer. This large differencein T_(on) was surprising and indicated that P05 is much more stable inthe citrate buffer compared with phosphate buffer. Accordingly, in someembodiments a formulation comprising an IL-1 inhibitor, e.g., P05,contains citrate buffer.

Example 13 Stability Studies

To test the stability of formulations described in Example 11,formulations with various concentrations of P05 were prepared (asdescribed in Example 11, supra) and were analyzed for baselinemeasurements. Vials of the formulated polypeptide were incubated at 25°C. for 0 days, 3 days, 1 week, 2 weeks, and 4 weeks and at 40° C. for 3days, 1 week, and 2 weeks. At least 2 vials were prepared per timepoint. Following incubation, the samples were analyzed using sizeexclusion HPLC (SE-HPLC), weak cation exchange HPLC (WCX-HPLC), reversedphase HPLC (RP-HPLC), concentration (A280−A320), visual appearance,formal inspection performed with photographs. The pH was analyzed at T=0and T=4 weeks. Osmolality was analyzed only at T=0. Visual inspectionand concentration were evaluated at 2 weeks and 4 weeks at both 25° C.and 40° C. All formulations were clear and colorless without visibleparticles after 2 weeks. The 25° C. samples were clear and colorlessafter 4 weeks. The results for these concentration studies areillustrated in Table 5.

TABLE 5 Initial Osmolality concentration at Concentration @ T = 25° C.Concentration @ T = 40° C. Sample (T = 0) mg/ml T = 0 2 weeks 4 weeks 2weeks 4 weeks C1 1.08 306 1.04 1.04 1.04 NA mOsm/kg C2 5.27 305 5.255.23 5.20 NA mOsm/kg C3 21.0 315 21.2 21.0 21.2 NA mOsm/kg

The pH of the 25° C. samples was determined at 4 weeks. In all cases,the pH at T=0 was 6.00-6.01 and the pH at 4 weeks was 6.03-6.07. Thesedata demonstrate stability of the formulations across a range ofconcentrations in the formulations at 25° C. for at least 4 weeks and at40° C. for at least 2 weeks.

SEC-HPLC

Purity was assessed using a size exclusion HPLC method using absorbanceand fluorescence detection. Briefly, Sepax Zenix® SEC-150 7.8 mm×20 cm(PN 213150-7820) columns were used. The mobile phase was 1×PBS.Evaluations were performed using an Agilent 1100 HPLC system inisocratic mode with a flow rate of 1 mL/minute, a total run time of 18minutes, at ambient temperature. Absorbance detection was at 280 nm withfluorescence detection at an excitation wavelength of 280 nm andemission detection wavelength of 350 nm. For experiments employingfluorescence detection of 1 and 5 mg/ml samples, 10 μg of polypeptideformulation was loaded and for absorbance detection of 20 mg/ml samples,50 μg of polypeptide formulation was loaded.

After 2 weeks of storage, the reference standard for the 1 mg/ml and 5mg/ml P05 formulations had an assayed purity of 99.1% and the purity of1 mg/ml and 5 mg/ml samples of the P05 formulation were 99.1%-99.2%,respectively, regardless of whether they were stored at 25° C. or 40° C.For the 20 mg/ml sample, the reference had a purity of 99.2%. After twoweeks of storage, the 20 mg/ml samples had a purity of 99.2%, regardlessof whether they were stored at 25° C. or 40° C. After 4 weeks of storageat 25° C., the 20 mg/ml P05 formulation had a purity of 99.2%.

wCEX-HPLC

In additional studies, a weak cation exchange HPLC method was used toassess the formulations. In this method, a Dionex ProPac® WCX-10, 4×250mm (PN 054993) column was used. Mobile phase A was 10 mM Na acetate, pH5.5 and mobile phase B was mobile A+0.25 M NaCl. The assay was performedusing an Agilent 1100 HPLC system with a flow rate of 1.2 mL/minute anda total run time of 35 minutes at ambient temperature. Detection wasperformed by assaying at 214 nm and 280 nm. Sample size was 25μg/injection. A summary of the results after storage for 2 weeks at 25°C. is shown in Table 6, after storage for 2 weeks at 40° C. in Table 7,and after storage for 4 weeks at 25° C. in Table 8.

The weak cation exchange assay is another method of assessing purity bymonitoring charge heterogeneity. Analysis of P05 formulation samplesresolves the main product peak from several product related impuritiesbased on charge. A typical preparation of P05 consists ofapproximately >85% main peak and several pre- and post-peaks. Pre-peak 1is unknown, Pre-peak 2 is a form of deamidated P05, Pre-peak 3 is a formof P05 with an N-terminal methionine. Post-peak 1 is a form of P05missing the N-terminal alanine, Post-peak-2 is a form of P05 missingboth the N-terminal alanine and proline, Post-peak 3 is unknown.

TABLE 6 25° C. Storage/2 weeks % % % % % % Pre-Peak Pre-Peak Pre-Peak %Post-Peak Post-Peak Post-Peak Total Form # Formulation 1 2 3 Main Peak 12 3 Area — Ref. Std. 0.0 1.0 1.3 91.5 5.5 0.3 0.3 1200 C1 01C6.0SP 0.01.4 1.3 90.3 6.1 0.4 0.4 1166 C2 05C6.0SP 0.0 1.1 1.1 91.5 5.6 0.4 0.41176 C3 20C6.0SP 0.0 1.5 1.3 90.8 5.4 0.5 0.5 1255

TABLE 7 40° C. Storage/2 weeks % % % % % % Pre-Peak Pre-Peak Pre-Peak %Post-Peak Post-Peak Post-Peak Total Form # Formulation 1 2 3 Main Peak 12 3 Area — Ref. Std. 0.0 1.1 1.4 91.4 5.5 0.4 0.2 1220 C1 01C6.0SP 0.01.9 1.3 89.4 6.5 0.6 0.3 1151 C2 05C6.0SP 0.0 1.8 1.4 89.3 6.5 0.7 0.31181 C3 20C6.0SP 0.0 2.0 1.4 89.5 6.1 0.7 0.3 1223

TABLE 8 25° C. Storage/ 4 weeks % % % % % % Pre-Peak Pre-Peak Pre-Peak %Post-Peak Post-Peak Post-Peak Total Form # Formulation 1 2 3 Main Peak 12 3 Area — Ref. Std. 0.0 1.1 1.4 91.4 5.5 0.4 0.2 1220 C1 01C6.0SP 0.01.9 1.3 89.4 6.5 0.6 0.3 1151 C2 05C6.0SP 0.0 1.8 1.4 89.3 6.5 0.7 0.31181 C3 20C6.0SP 0.0 2.0 1.4 89.5 6.1 0.7 0.3 1223

After 2 weeks of storage at 25° C., the percent main peak remainedsimilar to the reference for the 5 mg/ml formulation (C2), about 91%purity, whereas there was a slight decrease in purity for C3 (20 mg/ml)and C1 (1 mg/me as determined using this method. Decreases in the mainpeak and increases in pre-peak 2, post-peak 1, and post-peak 2 wereobserved for an samples following storage at 40° C. After storage forfour weeks, decreases were observed in the main peak purity and therewas an increase in pre-peak 2 and post-peak 1.

Reversed Phase HPLC (RP-HPLC)

Formulations were also assessed using RP-HPLC. The RP-HPLC assay isanother method of assessing purity by monitoring product heterogeneitybased on hydrophobicity. The method is capable of separating the nativemolecule from product related impurities that contain oxidizedmethionines. Pre-peaks 2 and 3 are oxidized forms of the P05, andpost-peaks 1 and 2 are acetylated forms of the molecule.

In this method, a Waters Symmetry® C4 (4.6×150 mm; 3.5 μm; PN 186000283)was used with a mobile phase A of 0.05% trifluoroacetic acid (TFA) inwater and mobile phase B was 0.05% TFA in 95% acetonitrile (ACN). Assayswere run using an Agilent 1200 HPLC system with a flow rate of 1mL/minute for a total run time of 35 minutes and a column temperature of55° C. Detection was performed at 280 nm. The amount of sample loadedfor 1 mg/ml and 5 mg/ml samples was 25 μg and the amount of sampleloaded was 50 μg. A summary of the results after storage for 2 weeks at25° C. is shown in Table 9, after storage for 2 weeks at 40° C. in Table10, and after storage at 4 weeks at 25° C. in Table 11 for the 1 mg/mland 5 mg/ml formulations. Data for the 20 mg/ml formulation after twoweeks of storage and 25° C. and 40° C. are shown in Tables 12 and 13,respectively.

TABLE 9 25° C. Storage for 2 Weeks % % % % % % Pre-Peak Pre-PeakPre-Peak Pre-Peak % Post-Peak Post-Peak Total Form. # Formulation 1 2 34 Main Peak 1 2 Area t = 2 week Ref. Std. 0.0 0.2 1.3 0.2 96.8 1.1 0.41650 C1 01C6.0SP 0.0 0.3 4.5 0.4 93.1 1.3 0.5 1602 C2 05C6.0SP 0.0 0.22.6 0.3 95.1 1.3 0.4 1602

TABLE 10 40° C. Storage for 2 Weeks, 1 mg/ml and 5 mg/ml Formulations %% % % % % Pre-Peak Pre-Peak Pre-Peak Pre-Peak % Post-Peak Post-PeakTotal Form. # Formulation 1 2 3 4 Main Peak 1 2 Area t = 2 week Ref.Std. 0.0 0.2 1.3 0.2 96.8 1.1 0.4 1650 C1 01C6.0SP 0.0 0.3 8.2 1.3 87.91.9 0.4 1571 C2 05C6.0SP 0.0 0.2 4.1 1.2 92.3 1.8 0.4 1591

TABLE 11 25° C. Storage for 4 Weeks, 1 mg/ml and 5 mg/ml Formulations %% % % % % Pre-Peak Pre-Peak Pre-Peak Pre-Peak % Post-Peak Post-PeakTotal Form. # Formulation 1 2 3 4 Main Peak 1 2 Area t = 4 weeks Ref.Std. 0.0 0.2 1.3 0.3 96.8 1.0 0.4 1648 C1 01C6.0SP 0.0 0.3 5.5 0.6 91.91.4 0.4 1583 C2 05C6.0SP 0.0 0.2 3.0 0.6 94.5 1.4 0.3 1598

TABLE 12 25° C. Storage for 2 Weeks, 20 mg/ml Formulation % % % % % %Pre-Peak Pre-Peak Pre-Peak Pre-Peak % Post-Peak Post-Peak Total Form. #Formulation 1 2 3 4 Main Peak 1 2 Area t = 2 week Ref. Std. 0.0 0.2 1.30.2 96.8 1.1 0.4 3297 C3 20C6.0SP 0.0 0.2 2.0 0.3 96.1 1.1 0.4 3340

TABLE 13 40° Storage for 2 Weeks, 20 mg/ml Formulation % % % % % %Pre-Peak Pre-Peak Pre-Peak Pre-Peak % Post-Peak Post-Peak Total Form. #Formulation 1 2 3 4 Main Peak 1 2 Area t = 2 week Ref. Std. 0.0 0.2 1.30.2 96.8 1.1 0.4 3297 C3 20C6.0SP 0.0 0.2 3.3 0.7 94.1 1.4 0.4 3270

After two weeks of storage at 25° C. or 40° C., a decrease in the mainpeak purity was observed and an increase in pre-peak 3 (oxidized P05)was observed for the 1 mg/ml and 5 mg/ml formulations. This effect wasmost prominent for the 1 mg/ml sample (C1) at high temperature. Afterfour weeks of storage at 25° C., a decrease in main peak purity and anincrease in pre-peak 3 (oxidized P05) was observed for theseformulations and this effect was most prominent for the 1 mg/ml sample.Interestingly, after two weeks of storage at 25° C., the 20 mg/mlformulation had a main peak percentage similar to T=0 (96.1% purity). Adecrease in the percent main peak and increase in pre-peak 3 (oxidizedP05) was observed for this sample after two weeks of storage at 40° C.After four weeks of storage at 25° C., the 20 mg/ml formulation showed aslight decrease in main peak purity and an increase in pre-peak 3(oxidized P05).

These analytical data demonstrate methods of analyzing the stability ofa chimeric cytokine formulation and in particular demonstrate thesurprising stability of a chimeric cytokine preparation comprising theP05 polypeptide. Accordingly, in some embodiments, the invention relatesto a formulation stored for at least 2 weeks, e.g., at least 4 weeks at25° C., e.g., 40° C. at a concentration of at least 1 mg/ml, at least 5mg/ml, or at least 20 mg/ml and has a purity of at least 92%, e.g., atleast 94%, or at least 96%.

Example 14 Stability of Formulations in Blow Fill Seal Containers

The process of packaging formulations into blow fill seal (BFS)containers involves plastic extrusion, molding, aseptic filling, andsealing in sequence. See, e.g., Liu, W. et al. 2011 BiopharmInternational, 24(7): 22-29. In the extrusion step, plastic granules aremelted at temperatures above 160° C. Subsequently, the plastic is moldedinto the desired container shape, filled with formulation solution, andhermetically sealed.

Because the plastic materials used to form the containers are gaspermeable to some degree, the stability of the formulation may sufferduring long term storage (e.g., due to evaporation of water from thecontainer and/or protein oxidation). Sealing such containers in analuminum foil pouch or other suitable package may protect theformulation from light-induced degradation. Sealing the containers insuch aluminum foil pouches with an inert gas, e.g., nitrogen or argoncan protect against oxidation. Accordingly, experiments were conductedto investigate the effects of packaging and subsequent storage of P05 inblow fill seal (BFS) containers, with or without aluminum foil poucheswith an inert gas overlay.

Testing was performed for formulations containing active pharmaceutical,P05. Bulk drug substance was formulated into an aqueous solutioncontaining 10 mM sodium citrate, 5% w/v sorbitol, 0.1% w/v poloxamer188, at pH 6.0 for blow fill processing. The target concentration forP05 was 5.0 mg/mL.

The formulation was cooled to about 2° C.-8° C. and filled intocontainers. Approximately 1000 containers were filled and the targetfill volume of the containers was 0.32 mL. A portion of the containerswere pouched in foil packages with a nitrogen overlay. An initialcharacterization was performed following the packaging into BFScontainers and further stability evaluations were conducted followingstorage at two temperatures (2° C. to 8° C. and 25° C.), with or withoutpouching.

The initial characterization analysis included: concentration by A₂₈₀,SDS-PAGE, SEC-HPLC, wCEX-HPLC, RP-HPLC, osmolality and particle analysisby light obscuration. The stability of P05 was monitored monthly bySEC-HPLC, wCEX-HPLC and RP-HPLC, with A₂₈₀ evaluation performed atmonths 4 and 5, and pH and osmolality at month 5.

Initial Characterization of P05 Formulation Following Blow FillProcessing Showed Retention of Stability

Initial analyses of P05 following blow fill processing demonstrated thatthe protein retained its chemical and physical stability despite theblow fill processing. The results from a P05 formulation not exposed tothe BFS process (aqueous formulation containing P05 at a concentrationof 50 mg/mL, 0.01% w/v poloxamer 188, 5% w/v sorbitol, 10 mM sodiumphosphate, at pH 6.5) were compared with a P05 formulation that wassubjected to the blow fill process (aqueous formulation containing P05at a concentration of 5 mg/mL, 0.1% w/v poloxamer 188; 5% w/v sorbitol;10 mM sodium citrate, at pH 6.0). These formulations were made with P05from the same production batch.

The results show that at time zero following the blow fill packaging,the blow filled formulation retained excellent stability even afterexposure to the potentially detrimental blow fill process, as indicatedby analyses using size exclusion chromatography, RP-HPLC, wCEX, andSDS-PAGE (see Table 15).

TABLE 15 Comparison of Stabilities P05 formulation not exposed to P05formulation after Analysis BFS process packaging in BFS container Size99% main peak 100% main peak Exclusion RP-HPLC 97% main peak 97.6% mainpeak wCEX 94% main peak 95.3% main peak 5% des-ala 4.2% des-ala + 0.2% +methionine species methionine species unresolved from main peak SDS-PAGEConforms to Conforms to reference reference *Measurements were averagedfrom three vials (pulled randomly) from the latter half of thefill/finishIn addition to the above assays, particle analysis was performed on theformulation packaged in BFS containers, using light obscurationaccording to method USP <789>. The subvisible particle counts werewithin the USP specifications for topical ophthalmics (having less thanor equal to 50 particles per ml for particles ≧10 μm and less than orequal to 5 particles per ml for particles ≧25 μm), consistent with thelack of visible precipitation in the BFS containers (data not shown).

These data demonstrate that P05 was both physically stable (according tomeasurements from SEC-HPLC, light obscuration, and visible observation)and chemically stable (according to measurements from wCEX-HPLC,RP-HPLC, SDS-PAGE) immediately following blow fill processing.

As part of the initial characterization, osmolality and concentration byA₂₈₀ were measured. The average osmolality measurement was 317 mOsm, andthe average concentration by A₂₈₀ was 5.0 mg/mL.

P05 Formulation Stored in Blow Fill Containers Retained Stability

The P05 formulation in BFS containers was stored in an incubator at 25°C. with 60% relative humidity or at 2 to 8° C. At monthly intervals,samples were analyzed by SEC, RP-HPLC, and wCEX-HPLC. At months 4 and 5,concentration was measured by A₂₈₀. At month 5, osmolality (to assessevaporation), pH and concentration by A₂₈₀ were also measured.

TABLE 16 SEC-HPLC Stability Results (% Main Peak) for P05 formulationStored in BFS containers 25° C./60% RH 2 to 8° C. Month Pouched NotPouched Pouched Not Pouched 0 100 100 100 100 2 99.0 99.0 99.2 99.3 399.7 99.8 99.8 99.8 4 99.3 99.4 99.5 99.5 5 99.6 99.7 99.8 99.9The SEC-HPLC results (see Table 16) indicate that the P05 formulationstored in BFS containers did not form aggregates at room temperature or2 to 8° C. for at least five months. Pouching of the vials afternitrogen flushing did not affect the physical stability of the productat either temperature.The wCEX-HPLC stability results for the P05 formulation are shown inTable 17 and Table 18 (% main peak and % deamidated peaks,respectively).

TABLE 17 wCEX-HPLC Stability Results (% Main Peak) for P05 formulationStored in BFS containers 25° C./60% RH 2 to 8° C. Month Pouched NotPouched Pouched Not Pouched 0 95.3 95.3 95.3 95.3 1 94.1 93.9 95.0 95.02 92.7 92.7 95.8 95.8 3 90.0 90.0 95.5 95.5 4 87.4 88.3 95.4 95.5 5 85.886.1 95.7 95.7

TABLE 18 wCEX-HPLC Stability Results (% Deamidated Peaks) for P05formulation Stored in BFS containers 25° C./60% RH 2 to 8° C. MonthPouched Not Pouched Pouched Not Pouched 0 0.8 0.8 0.8 0.8 1 1.6 1.8 1.21.2 2 2.7 2.7 0.12 0.50 3 4.7 4.8 0.42 0.41 4 6.7 7.0 0.60 0.51 5 9.58.5 0.34 0.33The wCEX-HPLC results indicate that the P05 formulation in blow-fillseal containers remained stable at room temperature for up to fivemonths. P05 also retained stability for at least 5 months at 2 to 8° C.Pouching of the containers after nitrogen flushing did not affect thestability of the product at either temperature.The RP-HPLC stability results for the P05 engineering run drug productare shown in Table 19 and Table 20 (% main peak and % oxidized proteinpeaks, respectively).

TABLE 19 RP-HPLC Stability Results (% Main Peak) for P05 formulationStored in BFS containers 25° C./60% RH 2 to 8° C. Month Pouched NotPouched Pouched Not Pouched 0 97.6 97.6 97.6 97.6 1 96.2 96.3 97.3 97.12 96.0 96.0 97.3 97.1 3 94.9 93.5 97.2 97.0 4 94.1 92.6 96.7 96.5 5 91.691.3 96.2 96.0

TABLE 20 RP-HPLC Stability Results (% Oxidized Peak) for P05 formulationStored in BFS containers 25° C./60% RH 2 to 8° C. Month Pouched NotPouched Pouched Not Pouched 0 2.2 2.2 2.2 2.2 1 3.3 3.7 2.4 2.5 2 3.64.1 2.4 2.6 3 4.6 5.1 2.5 2.7 4 5.4 6.4 2.9 3.1 5 8.4 7.4 3.6 3.5The RP-HPLC results indicate that the P05 formulation stored in BFScontainers was stable at room temperature and 2 to 8° C. for at leastfive months.Additionally, osmolality and pH measurements indicated that nosignificant change in osmolality or pH occurred over time, for samplesat 25° C. (see Table). This demonstrates that little to no evaporationoccurred, and that the pH of the solution remained stable. The proteinconcentration as assessed using A₂₈₀ was also consistent with previousmeasurements (see Table 21). Overall, EBI-005 exhibited excellentphysical stability after prolonged storage in blow fill seal vials at at2 to 8° C. and at ambient temperature (RT).

TABLE 21 Osmolality, pH, and Concentration for the P05 formulation afterFive Months of Storage in BFS Containers Osmo- Description TemperaturePackaging lality pH A₂₈₀ Active 25° C. Pouched 318 6.06 4.89Pharmaceutical 5 mg/mL Active 25° C. Not pouched 325 6.05 4.94Pharmaceutical 5 mg/mL Active  4° C. Pouched 320 6.11 4.87Pharmaceutical 5 mg/mL Active  4° C. Not pouched 319 6.07 4.87Pharmaceutical 5 mg/mL vehicle 25° C. Not pouched 328 6.02 0.00* vehicle 4° C. Not pouched 322 6.06 −0.02* *Blanked with Milli-Q ™ water

Example 15 Methionine Containing Formulations

In some embodiments, the invention relates to a formulation as describedherein containing methionine.

The use of antioxidant for P05 formulated in 10 mM sodium citrate, 5%w/v sorbitol, 0.1% w/v poloxamer 188, pH 6.0 was studied using twodifferent stress conditions: temperature (storage at 40° C.) and forcedoxidation using hydrogen peroxide. Hydrogen peroxide is a stressor thatcauses oxidation by free radicals and thus was used to emulate theoxidation effect that may occur after storage in multidose containersthat have been gamma irradiated (see below). P05 exhibits increases inoxidation levels when stored at high temperatures for prolonged periods.The use of either 10 mM methionine or 7 mM bisulfate added to theformulation was tested for the P05 formulation stored 3 weeks at 40° C.Additionally, the same formulation was tested using forced oxidation,where 10% v/v of 0.02% hydrogen peroxide was added to the samples. Thesamples were tested by RP-HPLC to assess the levels of oxidation. Table22 provides a data summary.

TABLE 22 RP-HPLC analysis of stressed samples with and withoutanti-oxidant % main % concentration Variable condition tested peakoxidized 1 mg/mL NA control (not stressed) 99 1 1 mg/mL no additive 3weeks at 40° C. 90 10 1 mg/mL +10 mM Methionine 3 weeks at 40° C. 98 2 1mg/mL +7 mM Bisulfate 3 weeks at 40° C. 88 12 20 mg/mL  no additive 3weeks at 40° C. 97 3 20 mg/mL  +10 mM Methionine 3 weeks at 40° C. 98 220 mg/mL  +7 mM Bisulfate 3 weeks at 40° C. 96 4 1 mg/mL no additiveperoxide added to 0.002% 38 62 1 mg/mL +10 mM Methionine peroxide addedto 0.002% 73 27 1 mg/mL +7 mM Bisulfate peroxide added to 0.002% 38 62These data demonstrate that methionine, but not bisulfate, reduces thelevel of oxidized protein, particularly at low concentrations forprotein stored at high temperature or with peroxide addition.

The effect of both container closure systems (blow fill and multi-dose),and the use of anti-oxidant in the formulation for multidose vials, werestudied for their effect on oxidation levels of P05. Multi-dosecontainers (vials) were sterilized by gamma irradiation Gammairradiation can result in the generation of free radicals in thecontainer that can be detrimental to the chemical stability of theprotein, specifically by oxidizing methionine residues on the molecule.Accordingly, the present experiment investigated whether the addition ofmethionine to P05 formulated in 10 mM sodium citrate, 5% w/v sorbitol,0.1% w/v poloxamer 188, pH 6.0 would ameliorate the oxidation of P05. 10mM methionine was used for P05 at 1 mg/mL in multi-dose or blow fillvials. The protein was stored at either 2-8° C. or ambient temperaturefor up to 4 weeks. The protein was analyzed by RP-HPLC to determinelevels of oxidized P05. Table 23 shows a summary of results.

TABLE 23 RP-HPLC analysis of P05 stored in either blow filled ormulti-dose containers ± methionine Container Additive Temperature Time(weeks) % Main Peak % Oxidized Peak blow fill none 2 to 8° C. 0 98.6 1.41 98   2   2 97.9 2.1 3 97.6 2.4 4 97.6 2.4 multidose none 2 to 8° C. 098.2 1.7 1 98.2 1.8 2 97.9 2.1 3 97.3 2.7 4 97.3 2.7 multidose 10 mMmethionine 2 to 8° C. 0 98.2 1.7 1 98.2 1.8 2 98.1 1.9 3 97.7 2.3 4 97.62.4 blow fill none ambient 0 98.6 1.4 1 97.3 2.7 2 96.6 3.4 3 96.5 3.5 4ND* ND* multidose none ambient 0 98.2 1.7 1 96.4 3.6 2 95.2 4.8 3 93.86.2 4 92.5 7.5 multidose 10 mM methionine ambient 0 98.2 1.7 1 96.9 3.12 96.2 3.8 3 95.3 4.7 4 95.3 4.7 *ND: not determined due to samplecontaminationAddition of methionine to the formulation reduced oxidation in themultidose container. For example, after 4 weeks of storage in multidosecontainers at ambient temperature, the % oxidized peak in theformulation without methionine was 7.5% and the % oxidized peak in theformulation with methionine was only 4.7%.

Example 16 Therapeutic Effects of EBI-005 Formulation and VehicleFormulation

A multicenter, double masked, randomized, placebo controlled clinicaltrial was completed to evaluate the safety and biological activity of anaqueous formulation of EBI-005 in patients with moderate to severe dryeye disease. The formulation employed in this study contained EBI-005(also referred to herein as P05) at a concentration of either 20 mg/mlor 5 mg/ml (see below), sodium carboxymethyl cellulose in aconcentration of 0.25% w/v; poloxamer 188 in a concentration of 0.1%w/v; sorbitol in a concentration of 5% w/v; and sodium phosphate in aconcentration of 10 mM. The trial was conducted in 74 patients at eightcenters in the United States. The trial was conducted in a naturalenvironment (a controlled adverse environment chamber was not used).

Patients were screened against eligibility criteria at a first visit.Patients who qualified for enrollment received topical administration ofvehicle in each eye three times per day for one week. At the conclusionof the one-week run-in period, patients were again reassessed againsteligibility criteria. Those patients who qualified under theseadditional criteria were randomized to one of three treatment groups.The Corneal Fluorescein Staining (CFS) score, Ocular Surface DiseaseIndex (OSDI) score and other measures taken at randomization arereferred to herein as baseline measures.

Eligible subjects were at least 18 years of age, with moderate to severedry eye disease. Additional eligibility criteria included the following:(i) OSDI score greater than or equal to 23 and less than 90 at the timeof screening; (ii) OSDI score greater than or equal to 19 atrandomization; (iii) CFS score greater than or equal to six and lessthan 15 on the NEI scale at the time of screening; and (iv) CFS scoregreater than or equal to five at randomization.

Patients who were randomized to a treatment group were treated in botheyes three times per day for six weeks beginning at randomization.Treatments for the three groups in this trial were as follows: (i) inthe first group, 22 patients received topical administration in each eyethree times per day of EBI-005 formulation containing EBI-005 at aconcentration of 20 mg/ml, (ii) in the second group, 22 patientsreceived topical administration in each eye three times per day ofEBI-005 formulation, containing EBI-005 at a concentration of 5 mg/ml,(iii) in the third group, 30 patients received topical administration ineach eye three times per day of vehicle formulation (vehicle formulationwas an aqueous formulation containing the same components as the EBI-005formulation, except that EBI-005 was not in the vehicle formulation).

Patients were assessed at screening; at randomization; at evaluationvisits on weeks two, four and six following randomization; and at afollow up visit one week after the completion of treatment. The timelinefor this clinical trial and number of patients randomized into theEBI-005 treatment and vehicle control groups are depicted in FIG. 7.Pain was assessed based on analysis of a single question from the 12questions of the OSDI that asked patients about painful or sore eyes.

Results are shown in FIGS. 8 to 10. These results show that signs andsymptoms of dry eye disease, as assessed using OSDI score (FIG. 8), pain(FIG. 9), and corneal fluorescein staining (CFS) score (FIG. 10),improved during the course of treatment with the EBI-005 formulations.Surprisingly, treatment with the vehicle only formulation also resultedin notable improvements in OSDI score, pain, and CFS score.

Example 17 A Double-Masked, Randomized, Controlled Study of EBI-005 (5mg/ml) Topical Ophthalmic Solution and Vehicle in Subjects with Moderateto Severe Dry Eye Disease (DED)

A study is conducted determining the efficacy of a vehicle formulation(10 mM sodium citrate, pH 6.0, 5% sorbitol (w/v), and 0.1% poloxamer 188(w/v)) and a therapeutic formulation (10 mM sodium citrate, pH 6.0, 5%sorbitol (w/v), and 0.1% poloxamer 188 (w/v) containing 5 mg/ml P05)given as a topical ophthalmic solution in each eye to subjects withmoderate to severe dry eye disease (DED) three times daily for 12 weeks.

Subjects are assessed for DED and inclusion criteria include having ahistory of dry eye disease (DED) in both eyes supported by a previousclinical diagnosis or have a self-reported history of subjectivecomplaints for at least 6 months prior to screening (Visit 1), haveongoing DED, in the same eye or both eyes, as defined by the followingcriteria at Visit 1: an OSDI score of ≧23 and ≦75 and have scored thepainful or sore eye question of the OSDI and a Total Corneal FluoresceinStaining Score of ≧6 (NEI scale) and <15. Screened subjects then undergoa five to eight day treatment with masked vehicle formulation, and arethen rescreened (Visit 2) to confirm they meet the randomizationcriteria at this visit. The randomization criteria include having atotal OSDI score of ≧19 and ≦50, having a total corneal fluoresceinstaining score of ≧5 (NEI scale) in the same qualifying eye as in Visit1 and CFS <15 in at least one eye, and having complied with the five toeight day masked vehicle formulation period. Compliance is defined asadministering at least 80% of the doses.

Subjects are then randomized to treatment with vehicle formulation ortherapeutic formulation and are provided with enough vehicle formulationor therapeutic formulation to administer one drop in each eye 3 timesdaily through visit 4 (week 3/note that the week numbering starts aftersubjects are randomized and assigned to receive vehicle or therapeuticformulation). Additional drug is dispensed at each subsequent visitthrough Visit 6 (week 9). Subjects are evaluated at Visit 3 (week 1),Visit 4 (week 3), Visit 5 (week 6), Visit 6 (week 9), Visit 7 (week 12)and Visit 8 (week 15). The last dose of study drug and final treatmentvisit are completed at Visit 7 (Week 12). The final evaluation is threeweeks later at Visit 8 (follow-up, Week 15).

Formulations are provided as a 2 to 8° C. solution in a low-densitypolyethylene (LDPE) blow fill unit. Subjects are provided with ReFreshPlus® tears to be used if required, over the 21 day period preceding thefinal evaluation. Subjects do not use any such artificial tears duringother parts of the study. Additional study information is available atclinicaltrials.gov, trial no. NCT01998802.

Evaluation of subjects during the study includes Total CornealFluorescein Staining (a sign), painful or sore eye question of the OSDIquestionnaire (a symptom), total OSDI and individual questions anddomains of the OSDI, inferior and central region CFS, global assessment(investigator and subject), subject-rated severity of individualsymptoms of dry eye, and Schirmer test without anesthesia. Improvementsin at least one of these criteria at the end of the first week (i.e.,after the three to eight day masked vehicle run-in) and/or duringsubsequent study visits for those subjects assigned to the vehicleformulation group compared to their initial evaluation, furtherdemonstrate the efficacy of a vehicle formulation. Improvements in atleast one of these criteria during subsequent study visits for thosesubjects assigned to the therapeutic formulation group compared to theirinitial evaluation or their evaluation after the three to eight daymasked vehicle run-in, further demonstrate the efficacy of a therapeuticformulation, e.g., a formulation comprising P05.

Other embodiments are within the scope of the following claims.

What is claimed is:
 1. An aqueous formulation comprising sodium citrate or sodium phosphate at a concentration of 8 to 12 mM; sorbitol at 4% to 6% (w/v); and poloxamer 188 at a concentration of 0.08% to 0.12% (w/v); wherein the formulation has a pH of 5.5 to 7.5 and wherein the formulation is effective for treating an ocular disorder.
 2. (canceled)
 3. The formulation of claim 1, wherein the formulation is substantially free of a therapeutic protein.
 4. The formulation of claim 1, wherein the formulation comprises sodium citrate at a concentration of 9 to 11 mM; sorbitol at 4.5 to 5.5% (w/v); and poloxamer 188 at a concentration of 0.09 to 0.11%. 5-6. (canceled)
 7. The formulation of claim 1, further comprising sodium carboxymethyl cellulose at a concentration of 0.1-1%.
 8. The aqueous formulation of claim 1, comprising sodium citrate at a concentration of 9-11 mM; sorbitol at 4.5-5.5% (w/v); and poloxamer 188 at a concentration of 0.09-0.11%, wherein the formulation has a pH of 5.7 to 6.3, wherein the formulation is substantially free of therapeutic protein, and wherein the formulation is effective for treating an ocular disorder.
 9. The formulation of claim 8, wherein the ocular disorder is dry eye disease. 10-11. (canceled)
 12. An aqueous formulation comprising 1-50 mg/ml of an IL-1β/IL-1Ra chimeric cytokine protein selected from P01, P02, P03, P04, P05, P06, or P07; a buffering agent selected from sodium citrate and sodium phosphate; sorbitol, e.g., at a concentration of 3.5-6.5% (w/v); and poloxamer 188, e.g., at a concentration of 0.07-0.13% (w/v); wherein the formulation has a pH of 5.5 to 7.5.
 13. The formulation of claim 12, wherein the chimeric cytokine protein is P05. 14-33. (canceled)
 34. The formulation of claim 1, wherein the formulation does not comprise a viscosity agent.
 35. The formulation of claim 12, wherein the formulation further comprises an amino acid.
 36. The formulation of claim 35, wherein the formulation comprises one or more of arginine, glutamic acid, histidine, and methionine.
 37. The formulation of claim 36, wherein methionine is present in the formulation at a concentration of 1 to 20 mM.
 38. The formulation of claim 37, wherein the formulation has reduced oxidation, compared to a corresponding formulation that does not comprise methionine, when the formulation is subjected to storage for at least 4 weeks at 25° C.
 39. The formulation of claim 38, wherein the formulation has reduced oxidation, compared to a corresponding formulation that does not comprise methionine, when the formulation is subjected to storage in a multidose container. 40-59. (canceled)
 60. The formulation of claim 12, wherein the formulation is packaged in a blow fill seal container. 61-63. (canceled)
 64. A method of treating an ocular disorder, the method comprising administering to a subject having the ocular disorder an aqueous formulation comprising sodium citrate or sodium phosphate at a concentration of 8 mM to 12 mM; sorbitol at 4% to 6% (w/v); and poloxamer 188 at a concentration of 0.08% to 0.12% (w/v); wherein the formulation has a pH of 5.5 to 7.5 and is substantially free of therapeutic protein, thereby treating the ocular disorder. 65-76. (canceled)
 77. A container or device comprising the formulation of claim
 1. 78. The container or device of claim 77, wherein the container or device has been stored at 25° C. for at least two weeks and the formulation is substantially free of particulates. 79-87. (canceled)
 88. The formulation of claim 8, further comprising carboxymethyl cellulose.
 89. The formulation of claim 12, further comprising carboxymethyl cellulose.
 90. The formulation of claim 12, wherein the formulation does not comprise a viscosity agent.
 91. A method of treating an ocular disorder, the method comprising administering to a subject having the ocular disorder the formulation of claim 12, thereby treating the ocular disorder.
 92. The container or device of claim 77, wherein the container or device comprises a multidose container.
 93. The container or device of claim 77, wherein the container or device comprises a blow fill seal container.
 94. A container or device comprising the formulation of claim
 12. 